JP2018528161A - Site-specific homogeneous complex with KSP inhibitor - Google Patents

Abstract

The present invention relates to site-specific homogeneous binder drug conjugates of kinesin spindle protein inhibitors; active metabolites of these conjugates; methods for producing these conjugates; use of these conjugates for the treatment and / or prevention of disease And the use of these conjugates for the manufacture of a medicament for the treatment and / or prevention of diseases, in particular hyperproliferative and / or angiogenic disorders, for example cancer diseases. Such treatment can be performed as a monotherapy or in combination with other medications or other therapeutic means. [Selection figure] None

Description

Introduction and State of the Art The present invention relates to site-specific homogeneous conjugate drug conjugates of kinesin spindle protein inhibitors, active metabolites of these conjugates, methods for producing these conjugates, these for the treatment and / or prevention of diseases. The use of the conjugates and the use of these conjugates for the manufacture of a medicament for the treatment and / or prevention of diseases, in particular hyperproliferative and / or angiogenic disorders, for example cancer diseases. Such treatment can be performed as a monotherapy or in combination with other medications or additional therapeutic means.

  Cancer diseases are the result of uncontrolled cell growth in the most diverse tissues. In many cases, new cells invade existing tissue (invasive growth) or they metastasize to distant organs. Cancer diseases occur in the most diverse organs and often follow the tissue-specific course of the disease. Therefore, the term “cancer” as a general name describes a large group of certain diseases of various organs, tissues and cell types.

  Early tumors may be able to be removed by surgical and radiotherapy means. Metastatic tumors are basically only symptomatically treatable by chemotherapy. The purpose in this case is to achieve the optimal combination of improved quality of life and extended life.

Complexes of conjugate proteins with one or more active compound molecules include antibody drug conjugates (ADCs) in which an internalizing antibody directed against a tumor-associated antigen is covalently bound to a cytotoxic drug via a linker. Known in form. Following introduction of the ADC into the tumor cell and then dissociation of the complex, the cytotoxic drug itself or cytotoxic metabolites formed therefrom are released within the tumor cell and exert their effects directly and selectively. be able to. In this way, in contrast to conventional chemotherapy, damage to normal tissue is limited to a fairly narrow range [see, eg, J. Org. M.M. Lambert, Curr. Opin. Pharmacol. 5 , 543-549 (2005); M.M. Wu and P.W. D. Senter, Nat. Biotechnol. 23 , 1137-1146 (2005); D. Center, Curr. Opin. Chem. Biol. 13 , 235-244 (2009); Docry and B.M. Stamp, Bioconjugate Chem. 21 , 5-13 (2010)]. Therefore, WO2012 / 171020 describes an ADC in which a plurality of poison molecules are bound to an antibody via a polymer linker. As possible poisons, WO2012 / 171020 includes in particular the substances SB743392, SB7159592 (Ispineci), MK-0371, AZD8477, AZ3146 and ARRY-520.

WO2012 / 171020

J. et al. M.M. Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005) A. M.M. Wu and P.W. D. Senter, Nat. Biotechnol. 23, 1137-1146 (2005) P. D. Center, Curr. Opin. Chem. Biol. 13, 235-244 (2009) L. Docry and B.M. Stamp, Bioconjugate Chem. 21, 5-13 (2010)

  The last substance listed is a kinesin spindle protein inhibitor. The kinesin spindle protein (also referred to as KSP, Eg5, HsEg5, KNSL1 or KIF11) is a kinesin-like motor protein that is essential for the function of the bipolar mitotic spindle. Inhibition of KSP leads to mitotic arrest leading to apoptosis over a relatively long period (Tao et al., Cancer Cell 2005 Jul 8 (1), 39-59). Following the discovery of the first cell-permeable KSP inhibitor, monastrol, KSP inhibitors have been established as a new class of chemotherapeutic agents (Mayer et al., Science 286: 971-974, 1999) and many patent applications It has become themes (eg WO 2006/044825; WO 2006/002236; WO 2005/051922; WO 2006/060737; WO 03/060064; WO 03/040979; and WO 03/049527). However, since KSP exerts its effect only during a relatively short period of mitosis, the KSP inhibitor must be present in a sufficiently high concentration in these initial phases.

Antibody conjugation methods typically involve chemical reactions with activated esters or maleimide functional groups of lysines or cysteines, respectively. However, these reactions are difficult to control with respect to site specificity and stereochemistry, resulting in heterogeneous products (Wang et al., Protein Sci. 14, 2436-2446 (2005); Hamblett et al., Clin. . Cancer Res 10, 7063-7070 (2004 );... Sun et al, Bioconjug Chem 16, 1282-1290 (2005);... Willner et al, Bioconjug Chem 4, 521-527 (1993)). As a result, heterogeneous ADCs can contain both unconjugated and excess antibodies. Unconjugated antibodies compete with drug-loaded species for antigen binding, which can reduce the activity of ADC therapy. On the other hand, high degree of antibody modification can result in antibody aggregation, increased toxicity, decreased stability and reduced half-life of ADCs in circulation (Sochaj et al., Biotechnology Advances, 33, 775-784 (2015)). It has been reported that heterogeneity of ADC species can affect its pharmacokinetics (PK), in vivo performance and safety profile (Jackson et al., PLo One 9, e83865 (2014); Junutula ... et al, Nat Biotechnol 26, 925-932 (2008);... Strop et al, Chem Biol 20, 161-167 (2013);.. Boswell et al, Bioconjugate Chem 22, 1994-2004 (2011 )). In addition, batch-to-batch consistency in ADC manufacturing is a difficult task and requires significant manufacturing capacity. Therefore, regulatory requirements may change in the future for new ADC approvals.

  Site-specific binding, where a known number of linker-drugs consistently bind to a given site, is one way to overcome these challenges. Non-uniformity is reduced, ADC characteristics are easier to predict, and composite production between batches is consistent. The drug: antibody ratio (DAR) is precisely controlled and can be adjusted to suit various linker-drugs. Various methods have been described in the literature for site-specific binding (Agarwal et al., Bioconjug. Chem. 26, 176-192 (2015); Cal et al., Angew. Chem. Int. Ed. Engl. 53, 10585-10877 (2014); Behrens et al., MAbs 6, 46-53 (2014); Panowski et al., MAbs 6, 34-45 (2014)). Examples of site-specific binding methods include enzyme methods using, for example, transglutaminases (TGases), glycyltransferases or formylglycine-generating enzymes (Sochaj et al., Biotechnology Advances, 33, 775-784). 2015).

  WO2014 / 198817 describes anti-TWEAKR antibodies and WO2015 / 189143 describes deglycosylated anti-TWEAKR antibodies, which can be used in antibody drug conjugates (ADCs). Furthermore, WO2015 / 096982 describes antibody drug conjugates (ADC) as kinesin spindle protein (KSP). In particular, this application describes ADCs with TWEAKR antibodies.

  The present invention provides site-specific homogeneity of a novel kinesin spindle protein inhibitor, in which the kinesin spindle protein inhibitor is bound to the glutamine side chain of the conjugate, and does not have the disadvantages of the random binding conjugate drug complex. A conjugate complex is provided. More specifically, the present invention uses transglutaminases (TGases), wherein the kinesin spindle protein inhibitor is bound to the glutamine side chain of the conjugate, and the above disadvantages of the random conjugate conjugate drug conjugate. Provided is a site-specific homogeneous conjugate complex of a novel kinesin spindle protein inhibitor that does not have

  Against this background, an object of the present invention is to provide a substance that can be beneficial in cancer therapy because it exerts an apoptotic effect after administration at a relatively low concentration.

  To achieve this object, the present invention provides a site-specific homogeneous complex of a conjugate or derivative thereof with one or more active compound molecules, wherein the active compound molecule binds to the conjugate via a linker L. One or more kinesin spindle protein inhibitors (KSP inhibitors) or complexes thereof are provided. The conjugate is preferably a conjugate protein or peptide, particularly preferably a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or an antigen-binding fragment thereof or an anti-EGFR antibody or it. An antigen-binding fragment of Particularly preferred is an anti-TWEAKR antibody that specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169), in particular anti-TWEAKR antibody TPP-2090, or anti-Her2 antibody trastuzumab. When the conjugate is an antibody, it preferentially contains an acceptor glutamine in the constant region. Such acceptor glutamines can be obtained by mutation to a suitable position of glutamine (eg mutation N297Q, Kabat EU numbering) or by formation of deglycosylated or deglycosylated antibodies (eg enzymatic deglycosylation with PNGase F). Alternatively, it can be introduced by Kabat EU numbering) by mutation of N297X. In the latter case of a deglycosylated or deglycosylated antibody, glutamine Q295 (Kabat EU numbering) becomes the acceptor glutamine. Highly preferred are antibodies comprising the mutation N297A or N297Q (Kabat EU numbering). Thus, in general, the antibodies described herein may be produced by deglycosylation of PNGase F or by heavy chain N297 (antibody Kabat numbering system, Kabat et al., Sequences of Proteins of Immunological, 5th Ed. Public Health. Also included are deglycosylated variants of these antibodies formed by mutations to any amino acid of Institutes of Health, Bethesda, MD (1991). Further, the antibodies described herein also include variants of the described antibodies that have been modified to include one or more acceptor glutamine residues for transglutaminase (TGase) catalysis.

One method of this binding is a literature approach that deals with site-specific binding of conjugates using transglutaminase. Transglutaminase (TGase), such as bacterial transglutaminase (BTG) (EC 2.3.2.13), is an enzyme that catalyzes the formation of a covalent bond between the γ-carbonylamide group of glutamines and the primary amine of lysines. It is a family. Some TGases also accept substrates other than lysine as amine donors, which are used to modify proteins, including antibodies, with suitable acceptor glutamine residues (Jeger et al., Angelwandte Chemie Int Ed. Engl 49 , 9995-9997 (2010); Josten et al., J. Immunol. Methods 240 , 47-54 (2000); Mindt et al., Bioconjugate Chem. 19 , 271-2; et al., in Antibiotic Drug Conjugates (Ducry, L., Ed.), pp 205-215, Humana Press. (2013)). On the other hand, transglutaminase is used to couple a drug to an antibody having a genetically artificial glutamine label, which is a transglutaminase acceptor glutamine introduced by genetic engineering (Strop et al., Chem. Biol. 2 0, 161-167 (2013 )). On the other hand, the conserved glutamine Q295 (the Kabat numbering system for IgGs) located in the constant region of the heavy chain is a single γ- for bacterial transglutaminase (EC 2.3.2.13) within the deglycosylated IgG1 backbone. It has been reported that no acceptor glutamine is present in the backbone in IgG1, which is a carbonylamide donor but glycosylated at heavy chain position N297 (kabat numbering) (Jeger et al., Angelwandte Chemie Int. Ed. Engl 49 , 9995-9997 (2010)). In summary, bacterial transglutaminase can be used to link the linker / drug amine group to the acceptor glutamine residue of the antibody. Such acceptor glutamine can be introduced by manipulation of the antibody by mutation or by formation of a deglycosylated antibody. Such deglycosylated antibodies may be formed by deglycosylation using N-glycosidase F (PNGase F) or by mutation of N297 (Kabat numbering) of the heavy chain glycosylation site to any other amino acid. Can do. Enzymatic conjugation of such deglycosylated antibodies may result in mutations N297D, N297Q (Jeger et al., Angelwandte Chemie Int. Ed. Engl 49, 9995-9997 (2010)), or N297S (patent applications WO20130392998A1 and WO2013093A). Reference is made to deglycosylated antibody variants. Enzymatic conjugation of such a deglycosylated antibody with transglutaminase provides an ADC with a DAR of generally 2 with both heavy chains functionalized site-specifically at position Q295 (Kabat numbering). . Antibody mutation N297Q provides another site for binding at each heavy chain, eg, both heavy chains are functionalized site-specifically at positions Q295 and Q297 (Kabat numbering). Resulting in an ADCS with 4 DARs. Antibody variants with mutations Q295N and N297Q provide a single acceptor glutamine residue at position Q297 (Simone Jeger, Site specific conjugation of tumor targeting ET). There are several examples in the literature describing site-specific binding of deglycosylated antibodies via transglutaminase (eg Denner et al., Bioconjugate Chemistry 19 , 569-578 (2014); Lhospice et al., Molecular Pharmaceuticals). 12 , 1863-1871 (2015)). The strategy using transglutaminase-catalyzed binding of deglycosylated antibody is summarized in FIG.

  The present inventors have found a method for achieving the above object by binding a conjugate to a KSP inhibitor in a site-specific and uniform manner. Furthermore, they have shown that transglutaminase can efficiently catalyze binding to deglycosylated antibody variants with the mutation N297A (Kabat numbering).

  The present invention relates to a site-specific homogeneous complex of a conjugate or a derivative thereof with one or more active compound molecules, wherein the active compound molecule is bound to the conjugate via a linker L. (KSP inhibitor), wherein the linker L is bound to a specific part of a conjugate, preferably a glutamine side chain of the conjugate.

  More specifically, the present invention is a site-specific homogeneous complex of a conjugate or derivative thereof with one or more active compound molecules, wherein the active compound molecule is bound to the conjugate via a linker L. It is a kinesin spindle protein inhibitor (KSP inhibitor), and provides a complex in which the linker L is bound to the glutamine side chain of the conjugate using transglutaminases (TGases).

The complex according to the present invention can be represented by the following general formula.

  BINDER represents a binder, preferably an antibody, L represents a linker, KSP represents a KSP inhibitor, m represents a number from 1 to 5, preferably 1, and n represents 2 to 10, preferably 2 To 4, preferably also 2 or 4. Where m is the number of KSP inhibitors per linker and n is the number of KSP inhibitors / linker complexes per BINDER. Therefore, the sum of all KSPs present in the complex is the product of m and n. Binder is preferably a binder peptide or protein, such as an antibody. When the binder is an antibody, it preferentially contains acceptor glutamine in the constant region. Such acceptor glutamines may be mutated to glutamine in a suitable position (eg, mutant N297Q, Kabat EU numbering) or by the formation of deglycosylated or deglycosylated antibodies (eg, enzymatic deglycosylation with PNGase F), Or by mutation of N297X, Kabat EU numbering). In the case of the latter deglycosylated or deglycosylated antibody, glutamine Q295 (Kabat EU numbering) becomes the acceptor glutamine. Highly preferred are antibodies comprising the mutation N297A or N297Q (Kabat EU numbering). Furthermore, the linker is attached to the glutamine residue of the binder peptide or protein or derivative thereof. Particularly preferred is binding to the glutamine residue of the antibody.

According to the present invention, the kinesin spindle protein inhibitor can have the following substructure I (sub):

Where
#A represents binding to the rest of the molecule;
R ^1a represents —H, —MOD, or — (CH ₂ ) _0-3 Z;
-MOD is represented as defined above,
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —SO ₃ H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C═O—NH—CHY). ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ;
R ^2a represents —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl;
R ^4a represents H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents —NHC (═O) —NH ₂ which may be substituted by linear or branched C _1-6 -alkyl, or aryl or benzyl which may be substituted by —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl,
Or R ^4a is R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P ₂ —NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O ) - group or cathepsin-cleavable group or a group ^R 21 of _{- (C = O) (0-1} ) - (P3) (0-2) -P2- represent,
R ²¹ represents C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _6-10. - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 -heteroalkoxy-, C _1-10 -alkyl-O—C _{6-10 -aryloxy-} , represents a C _5-10 -heterocyclic alkoxy group, which represents —NH ₂ , —NH-alkyl, —N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -SO 2 -N ( _{alkyl) 2} , May be substituted one or more times by —COOH, —C (═O) —NH ₂ , —C (═O) —N (alkyl) ₂ or —OH; or it may be —H or a group — (O) _x - represents a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 10,
R ²² is, -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent N An alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
Or R ^2a and R ^4a together represent —CH ₂ —CHR ¹⁰ — (with formation of a pyrrolidine ring) or —CHR ¹⁰ —CH ₂ —,
R ¹⁰ is, -H, halogen (preferably -F or _{-Cl), - NH 2, -COOH} , -SO 3 H, -SHC 1-4 - alkyl, halo _{-C 1-4} - _{alkyl, C 1- It represents 4} -alkoxy, hydroxy-substituted C _1-4 -alkyl, —C (═O) —O— (C _1-4 -alkyl) or —OH.

According to the present invention, the kinesin spindle protein inhibitor can be bound to the binder via a linker by replacement of a hydrogen atom with R ^1a , R ^2a , R ^4a or R ¹⁰ .

  The KSP inhibitor bound to this binder (or very often a KSP inhibitor because multiple KSP inhibitors are bound to the binder) is preferably a compound of formula (Ia) Salts, solvates, salts and epimers of solvates.

Formula (Ia):

Where
R ^1a represents —H, —MOD or — (CH ₂ ) _0-3 Z;
-MOD is represented as defined above,
Z represents -H, ^halogen, -OY ^{3, -SY} 3, the ^{-NHY 3, -C (O) -NY} 1 Y 2 or -C (O) -OY ^3,
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —SO ₃ H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH. -CHY ⁴⁾ represents the _1-3 COOH,
W represents -H or -OH;
Y ⁴ represents a linear or _branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ . Representation;
R ^2a represents —H, —MOD, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
-MOD is represented as defined above,
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl;
R ^4a represents —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl,
Or R ^4a is R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P ₂ —NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O ) -, or _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - group or a cathepsin cleavage, gender group, or a group of the formula _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2- represent,
R ²¹ represents C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _6-10. - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - heterocyclic alkoxy group _(which, -NH 2, -NH- alkyl, -N ( _{alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -S (=} O) 2 NH 2, -S (= O) ₂ -N (alkyl _{) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _{alkyl) 2,} or may be substituted one or more times by -OH), -. H or a group - _{(O) x} - represents a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is, -H, - alkyl (preferably _C 1 _{- 12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent N An alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
Or R ^2a and R ^4a together represent —CH ₂ —CHR ¹⁰ — (with formation of a pyrrolidine ring) or —CHR ¹⁰ —CH ₂ —,
R ¹⁰ is —H, halogen (preferably —F or —Cl), —NH ₂ , —COOH, —SO ₃ H, —SH, C _1-4 -alkyl, halo-C _1-4 -alkyl, C _{Represents 1-4} -alkoxy, hydroxy-substituted C _1-4 -alkyl, —C (═O) —O— (C _1-4 -alkyl) or —OH;
R ^3a is —MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl or heterocyclic alkyl group, preferably C _1-10 -alkyl, C _6-10 -aryl or C _{6- 10} -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, which represents 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 O-alkyl groups, 1 to 3 —SH groups, To 3 -S-alkyl groups, 1 to 3 -O-C (= O) -alkyl groups, 1 to 3 -O-C (= O) -NH-alkyl groups, 1 to 3 -NH-C = O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - alkyl group, one to three -S ( ═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 to 3 —NH ₂ group or 1 to 3 — (CH ₂ ) _0-3 optionally substituted by a Z group,
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
-MOD is represented as defined above,
n is 0, 1 or 2;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NHC (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) represents _0-3 Z ′,
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ^8a represents C _1-10 -alkyl or — (CH ₂ ) _0-2- (HZ ² );
HZ ² represents a 4 to 7 membered heterocyclic ring having 2 or less heteroatoms selected from the group consisting of N, O and S;
HZ represents a monocyclic or bicyclic heterocycle, which is one or more selected from the group consisting of halogen, C _1-10 -alkyl group, C _6-10 -aryl group and C _6-10 -aralkyl group Which may be substituted by a substituent, which may be substituted by a halogen;
-MOD as defined ^above, _{- (NR} 10) n - represents (G1) o -G2-G3,
R ¹⁰ represents —H; halogen or C ₁ -C ₃ -alkyl;
G1 represents —NHC (═O) —, —C (═O) —NH— or

(G1 is —NHC (═O) — or

R ¹⁰ does not represent NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y —, —C (═O) —, —CR ^x = N—O— may be interrupted one or more times,
The hydrocarbon group containing any side chain is replaced by —NHC (═O) —NH ₂ , —COOH, —OH, —NH ₂ , —NH—CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid. May have been
R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NHC (═O) —NH ₂ , —COOH, _{-OH, -NH 2, NH-CNNH} 2, sulfonamide, sulfone, optionally substituted by a sulfoxide or sulfonic acid may,
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl),
G3 represents -H or -COOH;
-MOD preferably has at least one group -COOH;
Said kinesin spindle protein inhibitor may be obtained by substitution of a hydrogen atom in R ^1a , R ^2a , R ^3a , R ^4a , R ^8a or R ¹⁰ , or optionally through one of the substituents of HZ, Is attached to the linker via R ^1a , R ^2a , R ^3a , R ^4a or R ¹⁰ .

  KSP-L- in the formulas shown above are preferably those having the following formula (IIa), and salts, solvates, solvate salts and epimers thereof.

Formula (IIa):

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or Represents CF, X ₂ represents C, X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH, X ₂ Represents N, X ₃ represents C,
(Preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N);
R ¹ is H, -L- # 1, -MOD or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
R ² is -L- # 1, H, -MOD, -C (= O) -CHY 4 -NHY 5 or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents —L— # 1, —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
Z is ^{-H, -OY} 3, -SY 3, represents -NHY 3, -C (= O) -NY 1 Y 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ⁴ represents R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) - or ^{_{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - group or a cathepsin cleavable group R _{^{21 - (C = O) (}} 0-1) - (P3) (1-2) -P2- represent,
R ²¹ is H, C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6- 10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - heterocyclic alkoxy group _(which, -NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (= O) 2 -N ( Alkyl) _{2 , -COOH, -C (= O)} -NH 2, -C (= O) -N ( _{alkyl) 2} or may be substituted one or more times by -OH,), - H or a group - (O) _x - represents a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids; when there are a plurality of amino acids P3, P3 can have the same or different amino acids as defined above;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents L- # 1, —H, —NH ₂ , —SO ₃ H, —COOH, —SH or —OH, and the hydrogen atom of the secondary amino group in the pyrrolidine ring is R ²¹ —C (═O _{) -P3 (0-2) -P2-NH} -CH (CH 2 C (= O) -NH 2) -C (= O) -SIG- may be replaced by;
L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
-SIG is a self-destructing group that gives a free secondary amine upon cleavage of the -C (= O) -SIG bond;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is -L- # 1, -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably -L- # 1 or C _1-10 -alkyl , C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl Group, 1 to 3 —SH group, 1 to 3 —S-alkyl group, 1 to 3 —O—C (═O) -alkyl group, 1 to 3 —O—C (═ O) -NH-alkyl group, Of three -NH-C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - group 1 to 3 —S (═O) ₂ —NH-alkyl groups, 1 to 3 —NH-alkyl groups, 1 to 3 —N (alkyl) ₂ groups, 1 to 3 —NH _Optionally substituted by ₂ or 1 to 3 — (CH ₂ ) _0-3 Z groups,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
n represents 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NHC (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) _0-3 represents Z '
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
R ⁵ is _{-L- # 1, -H, -NH 2} , -NO 2, halogen (especially _{-F, -Cl, -Br), -} CN, -CF 3, -OCF 3, -CH 2 F, Represents —CH ₂ F, —SH or — (CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
Z represents -H, -OY ³ , -SY ³ , halogen, -NHY ³ , -C (O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ are independently of each other —H, cyano, (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _2-10 -alkenyl, (fluorinated) C _2-10 -alkynyl, hydroxy, —NO ₂ , —NH ₂ , —COOH or halogen (especially —F, —Cl, —Br),
R ⁸ is (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _2-10 -alkenyl, (optionally fluorinated) C _2-10 -alkynyl. (may also be _{fluorinated) C 4-10} - represents (HZ ^2), - cycloalkyl or _{- (CH 2)} 0-2
HZ ² represents a 4 to 7 membered heterocycle having 2 or less heteroatoms selected from the group consisting of N, O and S, each of these groups being —OH, —COOH or —NH ₂ or — Optionally substituted by L- # 1;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
In -MOD definition above _{^- (NR} 10) n ^- represents (G1) o -G2-G3,
R ¹⁰ represents H or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is —NHC (═O) — or

, R ¹⁰ does not represent —NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y —, —C (═O) —, —CR ^x = N—O— may be interrupted one or more times,
The hydrocarbon chain including any side chain is replaced by —NH—C (═O) —NH ₂ , —COOH, —OH, —NH ₂ , NH—CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid. May have been
R ^y represents —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NH—C (═O) —NH ₂ , — _{COOH, -OH, -NH 2, NH} -CNNH 2, sulfonamide, sulfone, optionally substituted by a sulfoxide or sulfonic acid may,
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl;
G3 represents -H or -COOH;
-MOD preferably has at least one -COOH group.

  The complex according to the invention can have a chemically labile linker, an enzymatically labile linker or a stable linker. Particularly preferred are stable linkers and linkers cleavable by legumain or cathepsin.

  The present invention further provides a method for producing a site-specific homogeneous complex according to the present invention, as well as precursors and intermediates for its production.

  The production of the composite according to the invention usually comprises the following steps.

(I) Production of a linker precursor that may have a protecting group and has a reactive group capable of coupling to a binder;
(Ii) a derivative of a low molecular weight KSP inhibitor which may have a protecting group (preferably a KSP inhibitor having the substructure I (sub), particularly preferably of formula (Ia), in particular of formula (IIa) Obtaining a reactive KSP inhibitor / linker complex that may have a protecting group by attachment of the linker precursor to (in these formulas, there is still no linkage to the linker.);
(Iii) removal of protecting groups present in the KSP inhibitor / linker complex, and (iv) the binder according to the invention by site-specific binding of the binder to the KSP inhibitor / linker complex, preferably using transglutaminase / KSP inhibitor site-specific homogenous complex acquisition.

  Reactive group attachment can also be performed after construction of an optionally protected KSP inhibitor / linker precursor complex.

As explained above, attachment of the linker precursor to the low molecular weight KSP inhibitor is linked by R ^1a , R ^2a , R ^4a or R ¹⁰ in the substructure I (sub) and R in formula (Ia) by the linker. ^{1a, R 2a, R 3a,} R 4a, at ^{R 8a} or ^{R 10,} or by replacement of a hydrogen atom in ^{R 1, R 2, R 3} , R 4, R 5, R 8 or ^{R 10} in formula (IIa) Can happen. In the synthesis step prior to conjugation, the functional groups present may be present in protected form. Prior to the conjugation step, these protecting groups are removed by known methods of peptide chemistry. Complexing is performed chemically by various routes, as exemplarily shown in Schemes 2 to 6 in the Examples. In particular, a low molecular weight KSP inhibitor may be modified for attachment to a linker, for example by introduction of a protecting group or leaving group to facilitate substitution.

In particular, the present invention provides low molecular weight KSP inhibitors bound to a binder. These binder complexes are those having the following general formula (IIIa), and salts, solvates, solvate salts and epimers thereof.

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or CF X ₂ represents C, X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH, X ₂ represents N and X ₃ represents C (preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N);
R ¹ represents —H, —L-BINDER, —MOD or — (CH ₂ ) _0-3 Z;
Z is ^{-H, -NHY 3, -OY 3,} -SY 3, represents halogen, ^{-C (= O) -NY 1 Y} 2 or -C (= O) -OY ^3,
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ² represents -L-BINDER, H, -MOD, -C (= O) -CHY ⁴ -NHY ⁵ or-(CH ₂ ) _0-3 Z;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ is _L-# 1, represents _{-H, -NH 2, -SO 3 H} , -COOH, -SH, or -OH;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents -L-BINDER, -H, -C (O) -CHY ⁴ -NHY ⁵ or-(CH ₂ ) _0-3 Z;
Z is ^{-H, -OY} 3, -SY 3, represents -NHY 3, -C (= O) -NY 1 Y 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ⁴ represents R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) -, or ^{_{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) -, or a cathepsin cleavable group group, or a group of the formula _{^{R 21 - (C = O)}} (0-1) - (P3) (1-2) -P2- represent,
R ²¹ represents —H, C _1-10 -alkyl-, C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6. -10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy - , _{C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (= O) 2 -N (Al _{Le) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _alkyl) or with ₂ or -OH may be substituted one or more times, -H or a group -O _x - a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably C1-12- _alkyl), - _CH 2 -COOH, represents _-CH 2 _-CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ is _{L- # 1, -H, -NH 2} , -SO 3 H, -COOH, -SH, _{halogen, C 1-4 alkyl, C 1-4 haloalkyl, C 1-4} alkoxy, hydroxyl-substituted C _1-4 alkyl, COO (C _1-4 alkyl) or —OH; the hydrogen atom of the secondary amino group in the pyrrolidine ring is R ²¹ —C (═O) —P 3 _(0-2) —P 2 — _{NH-CH (CH 2 C (} = O) NH 2) -C (= O) -SIG- may be replaced by;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
-SIG is a self-destructing group that gives a free secondary amine upon cleavage of the -C (= O) -SIG bond;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is —L-BINDER, —MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably —L-BINDER, or C _1-10 -alkyl, _{Represents a} C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, It has 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl groups 1 to 3 —SH groups, 1 to 3 —S-alkyl groups, 1 to 3 —O—C (═O) -alkyl groups, 1 to 3 —O—C (═O ) − H-alkyl group, 1 to 3 —NH—C (═O) -alkyl group, 1 to 3 —NH—C (═O) —NH-alkyl group, 1 to 3 —S (═ O) _n -alkyl group, 1 to 3 —S (═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 May be substituted by 1 to 3 —NH ₂ groups or 1 to 3 — (CH ₂ ) _0-3 Z groups,
n is 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ is _{-H, - (CH 2) 0-3} -CH (NH-C (= O) -CH 3) Z ', - (CH 2) 0-3 -CH (NH 2) Z' or - ( CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
“Alkyl” preferably represents C _1-10 -alkyl);
R ⁵ is -L-BINDER, -H, -NH ₂ , -NO ₂ , halogen (especially F, Cl, Br), -CN, -CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F , -SH or - it represents _{(CH 2) 0-3} Z,
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁸ is C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _2-10 -alkynyl, fluoro-C _2-10 -alkynyl , C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl or — (CH ₂ ) _0-2- (HZ ² ),
HZ ² represents a 4 to 7 membered heterocyclic ring (preferably oxetane) having 2 or less heteroatoms selected from the group consisting of N, O and S, each of these groups being —OH, —COOH or It may be substituted by -NH ₂ or -L-BINDER;
L represents a linker;
BINDER represents a binder or a derivative thereof, and the binder may be bound to a plurality of active compound molecules,
One representative of R ¹ , R ² , R ³ R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents -L-binder;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} - represents alkynyl, _hydroxy, -NO _2, -NH 2, -COOH, or halogen (especially -F, -Cl, -Br), and - alkynyl, fluoro _{-C 2-10}
-MOD is ^- represents (G1) o -G2-G3, - (NR 10) n
R ¹⁰ represents —H or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is —NH—C (═O) — or

R ¹⁰ does not represent NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y — may be interrupted one or more times by one or more, and R ^y is —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ - alkenyl or _C 2 _{-C 10} - alkynyl, _each of -NH-C (= O) -NH 2, -COOH, -OH, -NH 2, NH-CN-NH 2, sulfonamide, sulfone, Optionally substituted by sulfoxide or sulfonic acid, or -C (= O)-, -C ^x = N-O-a represents, ^{R x} is _-H, C 1 -C ₃ - alkyl or phenyl,
Hydrocarbon chain containing either side _{chains, -NH-C (= O)} -NH 2, -COOH, -OH, -NH 2, NH-CN-NH 2, sulfonamide, sulfone, sulfoxide or sulfone acid May be replaced by
G3 represents -H or -COOH;
-MOD preferably has at least one group -COOH.

  FIG. 1 shows the sequences of TWEAKR cysteine-rich domains (amino acids 34 to 68) of various species (numbers indicate signal sequences; amino acid positions in the full-length construct including SEQ ID NO: 169).

  FIG. 2A is a schematic diagram of the structure of TWEAKR (SEQ ID NO: 169). The figure shows an extracellular domain (amino acids 28 to 80) (SEQ ID NO: 168), a transmembrane domain-TM (81 to 101) and an intracellular domain (102 to 129) containing a cysteine-rich domain (36 to 67). ing. TPP-2202—the complete extracellular domain (28-80) is fused to the FIgG domain of hIgG1. The extracellular domain with TPP-2203-N-terminal and C-terminal truncations (34 to 68) is fused to the FIgG domain of hIgG1. The disulfide bridges Cys36-Cys49, Cys52-Cys67 and Cys55-Cys64 are indicated by black bars. The N-terminal and C-terminal TPP-2203 contain two amino acids and one amino acid, respectively, in comparison with the unmodified cysteine-rich domain, thereby ensuring proper folding. The extracellular domain (28-68) with TPP-1984-C terminal truncation is fused to the HIS6 label. All three constructs show equivalent binding to the antibody according to the invention and PDL-192 (TPP-1104). P4A8 (TPP-1324) binds only to the full length extracellular domain (TPP-2202).

  FIG. 2B is the amino acid sequence of the extracellular domain: amino acid 64 is essential for TWEAK ligand binding; and amino acid 47 is essential for binding of an antibody according to the invention as confirmed herein. Has been.

  FIG. 3 shows the interaction of the TWEAKR extracellular domain antibody with a reference antibody. Shown are TWEAKR-Fc fusion protein coating (TPP-2202, 1 μg / mL) and 0.08 μg / mL (open bar) and 0.03 μ / mL (black bar) soluble binding partners Results of ELISA with biotinylated IgG as Detection was performed using streptavidin-HRP and Amplex Red substrate. Y is “ELISA signal intensity [Rfu]”; X is “test antibody construct”: a is “TPP-2090”; b is “TPP-2084”; c is “PDL-192 ( D is “P4A8 (TPP-1324)”; e is “P3G5 (TPP-2195)”; f is “136.1 (TPP-2194)”; h Is “ITEM1”; i is “ITEM4”; j is a mice isotype control; k is a human isotype control. All antibodies examined show saturation binding at a concentration of 80 ng / mL.

  FIG. 4 is the interaction of the TWEAKR cysteine-rich domain with an antibody according to the invention and a reference antibody. Shown are TWEAKR (34-68) -Fc fusion protein coating (TPP-2203, 1 μg / mL) and 0.08 μg / mL (open bar) and 0.3 μ / mL (black bar) Results of ELISA with biotinylated IgG as a soluble binding partner. Detection was performed using streptavidin-HRP and Amplex Red substrate. X is a “test antibody construct”: a is “TPP-2090”; b is “TPP-2084”; c is “PDL-192 (TPP-1104)”; d is “P4A8 ( E is “P3G5 (TPP-2195)”; f is “136.1 (TPP-2194)”; h is “ITEM1”; i is “ITEM4” Yes; j is a misisotype control; k is a human isotype control. All antibodies examined show saturation binding at a concentration of 80 ng / mL.

  FIG. 5 is the interaction of TWEAKR (28-68) with an antibody according to the invention and a reference antibody. Shown are TWEAKR (28-68) -HIS coating (TTP-1984, 1 μg / mL) and 0.08 μg / mL (open bar) and 0.3 μ / mL (black bar) soluble Results of ELISA with biotinylated IgG as binding partner. Detection was performed using streptavidin-HRP and Amplex Red substrate. X is a “test antibody construct”: a is “TPP-2090”; b is “TPP-2084”; c is “PDL-192 (TPP-1104)”; d is “P4A8 ( E is “P3G5 (TPP-2195)”; f is “136.1 (TPP-2194)”; h is “ITEM1”; i is “ITEM4” Yes; j is a misisotype control; k is a human isotype control. All antibodies examined show saturation binding at a concentration of 80 ng / mL.

  FIG. 6A is an alanine scan of a cysteine-rich domain. TWEAKR (34-68) -Fc muteins were analyzed for PDL-192 (TPP-1104) (X)-and TPP-2090 (Y) -binding. S37A, R38A, S40A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A and L65A muteins were expressed in HEK293 cells (black diamonds). PFL192 (TPP-1104) and TPP-2090 were coated (1 μg / mL), and 8-fold diluted supernatant of HEK293 fermentation broth was added for TWEAKR protein binding. X is the ELISA strength [Rfu] of the “PDL-192 / TTP-1104) interaction, and Y is the ELISA strength [Rfu] of the TPP-2090 interaction. TPP-2090 (Y) shows decreased binding of the D74A-TWEAKR mutein (closed box) and PDL-192 (TPP-1104) (X) shows decreased binding to R56A (dotted line) Box).

  BY is “binding in% [%] normalized to wild type binding signal”, 1 is “TPP-2090”; 2 is “PDL-192 (TPP-1104)” Yes; 3 is “P4A8 (TPP-1324)” (1 μg / mL), TWEAKR mutant was added at 250 ng / mL, and detection was by anti-HIS HRP. Compared to the wild type construct, TTP-2090 shows less than 5% binding.

  FIG. 7 is an NMR structure of the TWEAKR extracellular domain published by Pellegrini et al. (FEBS280: 1818-1829). TWEAK binding is dependent on L46 (Pellegrini et al.), TTP-2090 binding is dependent on and PDL-192 binds to R56. PDL-192 binds opposite to the TWEAK ligand binding site and TPP-2090 binds directly to the TWEAK ligand site.

  FIG. 8 summarizes the strategy using transglutaminase-catalyzed complexation of deglycosylated antibodies.

The sequence of TWEAKR cysteine-rich domains (amino acids 34 to 68) of various species (numbers indicate signal sequences; amino acid positions in full-length constructs including SEQ ID NO: 169). It is a schematic diagram of the structure of TWEAKR (SEQ ID NO: 169). Amino acid sequence of the extracellular domain. Interaction of the TWEAKR extracellular domain antibody with a reference antibody. FIG. 2 is an interaction of a cysteine-rich domain of TWEAKR with an antibody according to the invention and a reference antibody. TWEAKR (28-68) interaction with an antibody according to the invention and a reference antibody. Alanine scan of cysteine-rich domain. It is a figure which shows the binding [%] in% unit normalized with respect to the wild type binding signal. It is the NMR structure of the TWEAKR extracellular domain published by Pellegrini et al. (FEBS280: 1818-1829). Summarizes strategies using transglutaminase-catalyzed complexation of deglycosylated antibodies.

  The present invention relates to a site-specific homogeneous complex of a binder or derivative thereof with one or more active compound molecules, wherein the active compound molecule binds to the binder via a linker L (KSP). Inhibitors), wherein the linker L is bound to a specific site of the binder, preferably a glutamine side chain of the binder.

  More specifically, the present invention relates to a site-specific homogeneous complex of a binder or a derivative thereof with one or more active compound molecules, wherein the active compound molecule is bound to the binder via a linker L. Provided is a protein inhibitor (KSP inhibitor), wherein the linker L is bound to a glutamine side chain of a binder using transglutaminases (TGases).

The complex according to the present invention can be represented by the following general formula.

  BINDER represents a binder, preferably an antibody, L represents a linker, KSP represents a KSP inhibitor, m represents a number from 1 to 5, preferably 1, and n represents 2 to 10, preferably 2 To 4, preferably also 2 or 4. Where m is the number of KSP inhibitors per linker and n is the number of KSP inhibitors / linker complexes per BINDER. Therefore, the sum of all KSPs present in the complex is the product of m and n. KSP-L preferably has the formula (IIa) shown above. Binder is preferably a binder peptide or protein, such as an antibody. When the binder is an antibody, it preferentially contains acceptor glutamine in the constant region. Such acceptor glutamines may be mutated to glutamine in a suitable position (eg, mutant N297Q, Kabat EU numbering) or by the formation of deglycosylated or deglycosylated antibodies (eg, enzymatic deglycosylation with PNGase F), Or by mutation of N297X, Kabat EU numbering). In the case of the latter deglycosylated or deglycosylated antibody, glutamine Q295 (Kabat EU numbering) becomes the acceptor glutamine. Highly preferred are antibodies comprising the mutation N297A or N297Q (Kabat EU numbering). Furthermore, the linker is attached to the glutamine residue of the binder peptide or protein or derivative thereof. Particularly preferred is binding to the glutamine residue of the antibody.

  The binders that can be used according to the invention, the KSP inhibitors that can be used according to the invention and the linkers that can be used in combination without limitation are described below. In particular, the binder represented in each case as preferred or particularly preferred is combined with the KSP inhibitor represented in each case as preferred or particularly preferred, and is represented in each case as preferred or particularly preferred as appropriate. It can be used in combination with a linker.

KSP inhibitors and their binder complexes Low molecular weight KSP inhibitors are known, for example from WO 2006/044825; WO 2006/002236; WO 2005/051922; WO 2006/060737; WO 03/060064; WO 03/040979; and WO 03/049527. .

Basically, a KSP inhibitor has the following substructure I (sub):

Where
#A represents binding to the rest of the molecule;
R ^1a represents —H or — (CH ₂ ) _0-3 Z;
Z represents —H, halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —SO ₃ H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH. -CHY ⁴⁾ represents the _1-3 COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ Represents;
R ^2a represents —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl;
R ^4a represents H, —C (O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents —NH—C (═O) —NH ₂ which may be substituted by linear or branched C _1-6 -alkyl, or aryl or benzyl which may be substituted by —NH ₂ Represents
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl,
Or R ^4a is R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P ₂ —NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O ) - group or cathepsin-cleavable group, or a group of the formula ^R 21 a _{- (C = O) (0-1} ) - (P3) (0-2) -P2- represent,
R ²¹ represents —H, C _1-10 -alkyl-, C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C. _6-10 - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy _{-, C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl , -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (= O) ₂ -N (A _{Kill) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _alkyl) may be substituted one or more times with ₂ or -OH; or it, -H or a group - _(O) x - represents a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is, -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent N An alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
Or R ^2a and R ^4a together represent —CH ₂ —CHR ¹⁰ — (with formation of a pyrrolidine ring) or —CHR ¹⁰ —CH ₂ —,
R ¹⁰ is, -H, halogen (preferably -F or _{-Cl), - NH 2, -COOH} , -SO 3 H, -SHC 1-4 - alkyl, halo _{-C 1-4} - _{alkyl, C 1- It represents 4} -alkoxy, hydroxy-substituted C _1-4 -alkyl, —C (═O) —O— (C _1-4 -alkyl) or —OH.

The particularly high frequency is the following substructure II (sub).

Where
#A, R ^1a , R ^2a and R ^4a have the same meaning as in I (sub), R ^12a and R ^13a represent —H,
Or R ^12a and ^{R 13a} is (form a piperidine _{ring) together,} - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents —H, —NH ₂ , —COOH, —SO ₃ H, —SH or —OH;
Z is ^{-H, -OY} 3, -SY 3, represents -NHY 3, -C (= O) -NY 1 Y 2 or -C (= O) -OY ^3,
Y ¹ and Y ² are independently of each other —H, —NH ₂ or — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. )
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ , —COOH or — (C═O) —NH—CHY ⁴ ) _1-3 COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ .

In particular, many KSP inhibitors have the substructure II (sub) and R ^1a , R ^2a , R ^4a , R ^12a and R ^13a represent H.

  According to the present invention, a KSP inhibitor of substructure I (sub) or substructure II (sub) can be used. KSP inhibitors used according to the present invention also include, for example, ispinesive (Cytokinetics / GSK), MK-0731 (Merck), AZD4877 (AstraZeneca), ARRY-520 (Array BioPharmacia) and ARQ621 (ArQule).

  Preferred KSP inhibitors according to the present invention are those having the following basic structure, and salts, solvates, solvate salts and epimers thereof:

Formula (Ia):

Where
R ^1a represents —H, —MOD or — (CH ₂ ) _0-3 Z;
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —SO ₃ H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH. -CHY ⁴⁾ represents the _1-3 COOH,
W represents -H or -OH;
Y ⁴ represents a linear or _branched C _1-6 -alkyl which may be substituted by —NH—C (═O) —NH ₂ , an aryl which may be substituted by —NH ₂ or Represents benzyl;
R ^2a represents —H, —MOD, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl;
R ^4a represents —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl,
Or R ^4a is R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P ₂ —NH—CH (CH ₂ —C (═O) —NH ₂ ) —C (= O) -, or _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - group or a cathepsin, cleavable group, or a group of the formula _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2- represent,
R ²¹ represents —H, C _1-10 -alkyl-, C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C. _6-10 - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy _{-, C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - heterocyclic alkoxy groups _(which, -NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (= O) 2 -N (Alky _{) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _{alkyl) 2,} or may be substituted one or more times by -OH.) Represent, or -H Or the group — (O) _x — (CH ₂ CH ₂ O) _y —R ²² ,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is, -H, - alkyl (preferably _C 1 _{- 12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent N An alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
Or R ^2a and R ^4a together represent —CH ₂ —CHR ¹⁰ — (with formation of a pyrrolidine ring) or —CHR ¹⁰ —CH ₂ —,
R ¹⁰ represents —H, halogen (preferably —F or —Cl), —NH ₂ , —COOH, —SO ₃ H, —SH or —OH, and the hydrogen atom of the secondary amino group in the pyrrolidine ring is R ²¹ —C (═O) —P 3 _(0-2) —P ₂ —NH—CH (CH ₂ C (═O) NH ₂ ) —C (═O) —SIG— may be substituted;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent N An alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
-SIG is a self-destructing group that provides a free secondary amine upon cleavage of the -C (= O) -SIG bond;
R ^3a is —MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl or heterocyclic alkyl group, preferably C _1-10 -alkyl, C _6-10 -aryl or C _{6- 10} -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, which represents 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 O-alkyl groups, 1 to 3 —SH groups, To 3 -S-alkyl groups, 1 to 3 -O-C (= O) -alkyl groups, 1 to 3 -O-C (= O) -NH-alkyl groups, 1 to 3 -NH-C = O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - alkyl group, one to three -S ( ═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 to 3 —NH ₂ group or 1 to 3 — (CH ₂ ) _0-3 optionally substituted by a Z group,
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
-MOD is represented as defined above,
n is 0, 1 or 2;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NHC (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) represents _0-3 Z ′,
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ^8a represents C _1-10 -alkyl,
HZ represents a monocyclic or bicyclic heterocycle, which is one or more selected from the group consisting of halogen, C _1-10 -alkyl group, C _6-10 -aryl group and C _6-10 -aralkyl group Which may be substituted by a substituent, which may be substituted by a halogen;
-MOD as defined ^above, _{- (NR} 10) n - represents (G1) o -G2-G3,
R ¹⁰ represents —H; halogen or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is -HC (= O)-or

R ¹⁰ does not represent NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y —, —C (═O) —, —CR ^x = N—O— may be interrupted one or more times,
The hydrocarbon group containing any side chain is replaced by —NHC (═O) —NH ₂ , —COOH, —OH, —NH ₂ , —NH—CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid. May have been
R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NHC (═O) —NH ₂ , —COOH, _{-OH, -NH 2, NH-CNNH} 2, sulfonamide, sulfone, optionally substituted by a sulfoxide or sulfonic acid may,
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl),
G3 represents -H or -COOH;
-MOD preferably has at least one group -COOH.

According to the present invention, such a kinesin spindle protein inhibitor can be obtained by substituting a hydrogen atom in R ^1a , R ^2a , R ^3a , R ^4a , R ^8a or R ¹⁰ , or optionally among the substituents of HZ Can be linked to the linker via one of the following, in particular via R ^1a , R ^2a , R ^3a , R ^4a or R ¹⁰ .

  The substituents of formula (Ia) preferably have the following meanings, and these preferred meanings are preferably combined with one another.

R ^1a preferably represents H or — (CH ₂ ) _0-3 Z;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —SO ₃ H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
R ^2a and R ^4a independently of one another, preferably represent —H, —L— # 1, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z, or R ^2a and ^{R 4a} together form (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents —H, —SO ₃ H, —NH ₂ , —COOH, —SH or —OH;
Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
Y ⁴ represents, independently of each other, linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or may be substituted with —NH ₂ . Represents aryl or benzyl,
Y ⁵ represents H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl.

^{Alternatively, R 4a _{is, R 21 - (C = O}} ) (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 C (= O) -NH 2) -C (= O) - or _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - or the formula ^{R 21} - ( C = O) _(0-1) -(P3) _(1-2) -P2- can be a cathepsin-cleavable group. In another option, the —NH hydrogen atom on the pyrrolidine ring is R ²¹ —C (═O) —P 3 —P ₂ —NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O). Replaced by -SIG-.

Group ^{_{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 C (= O) -NH 2) -C (= O) -, R ^{_{21 - (C = O) (}} 0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - or ^R 21 -C (= O) -P3 _{-P2-NH-CH (CH 2} C (= O) -NH 2) -C (= O) -SIG- are believed to cleave in vivo by enzymatic legumain. Therefore, in the following, these groups are referred to as “legumain cleavable groups”. Legumain cleavable group of the formula _{- (C = O) (0-1} ) - (P3) (0-2) -P2-NH-CH (CH 2 C (= O) -NH 2) -C (= O ) - or _{- (C = O) (0-1} ) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - with a. In conjugates of the present invention, this group is preferably of the formula ^{_{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 C (= O) —NH ₂ ) —C (═O) #, ie a legumain cleavable group has a group R ²¹ at one end and at the other end (— #) it is attached to the amino group equivalent position R ^4a in formula Ia doing.

—NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) — (ie, asparagine) and —NH—CH (CH ₂ COOH) —C (═O) — (ie, aspartic acid ) Exists in the natural L configuration. Legumain cleavable group, in addition to asparagine, one to three additional amino acid _{(-P2-NH-CH (CH} 2 C (= O) -NH 2) -C (= O) -; - P3-P2 _{-NH-CH (CH 2 C (} = O) -NH 2) -C (= O) - ;-( P3) (2) -P2-NH-CH (CH 2 C (= O) -NH 2) - because it contains a), di - - C (= O), tri - or tetra peptide or derivatives thereof _{(dipeptides: -P2-NH-CH (CH} 2 C (= O) -NH 2) -C (= O) -; _{tripeptide: -P3-P2-NH-CH} (CH 2 C (= O) NH 2) -C (= O) -; tetrapeptide _{:-( P3) 2 -P2-NH-} CH _{(CH 2 C (= O)} -NH 2) -C (= O) -). The same is true when the legumain cleavable group contains aspartic acid.

  P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His Preferably an amino acid selected from Ala, Gly, Val, Leu, Ile, Pro, Ser, Thr, citrulline and Asn. P2 is usually present in the natural L configuration. Particularly preferred is L-Ala.

  P3 is independently Gly, Pro, Ala, Val, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline, or N- thereof. An amino acid selected from alkyl-amino acids, preferably N-methyl-amino acids. P3 is preferably selected from His, Pro, Ala, Val, Leu, Ile, Gly, Ser, citrulline and Gln. P3 is usually present in the natural L configuration. Particularly preferred is L-Ala.

Particularly preferred legumain cleavable group is -L-Ala-L-Ala- L-Asn- ( e.g. ^{R 21 -L-Ala-L-} Ala-L-Asn- #).

R ²¹ is preferably —H, C _1-5 -alkyl, C _5-10 -aralkyl, C _1-5 -alkoxy, C _6-10 -aryloxy, C _5-10 -heteroalkyl-, C _{5- 10} -heterocyclic alkyl-, heteroaryl-, heteroaryl-alkyl-, C _5-10 -heteroalkoxy-, or C _5-10 -heterocyclic alkoxy groups, which are —COOH, —C (═ O) —Oalkyl, —C (═O) —O—NH ₂ , —NH ₂ or —N (alkyl) ₂ ,) may be substituted one or more times; or the group —O _x — (CH ₂ It represents CH _{2 O)} y ^{-R 22,}
x is 0 or 1,
v is a number from 1 to 10,
R ²² is -H, - alkyl (preferably _{C 1-12} - _alkyl), - represents _{CH 2 -COOH, -CH 2 -CH 2} -COOH, or _{-CH 2 -CH} 2 -NH 2.

“Alkyl” means herein an alkyl group having up to 20 carbon atoms, preferably C _1-12 -alkyl.

The cathepsin cleavable group has the formula-(C = O) _(0-1) -(P3) _(1-2) -P2-. In conjugates of the present invention, this group is preferably of the formula _{^{R 21 - (C = O)}} (0-1) - (P3) (1-2) -P2- # having, i.e. cathepsin cleavable group has one end has a group ^{R 21,} the other end - in (#), which is bonded to an amino group equivalent position ^{R 4a} in formula Ia. Here, R ²¹ , P2 and P3 have the same meaning as in the legumain cleavable group. Particularly preferred cathepsin cleavable groups are those in which P2 is selected from alanine, lysine and citrulline and P3 is selected from valine, alanine and phenylalanine, in particular the formula R ^21- (C = O) _(0-1) -P3-P2-.

R ³ is preferably optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group,-L-# 1, -MOD or _{C 1-10,} - _{alkyl, C 6-10} - aryl Or a C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, which has 1 to 3 -OH group, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 O-alkyl groups, 1 to 3- SH group, 1 to 3 —S-alkyl group, 1 to 3 —O—C (O) -alkyl group, 1 to 3 —O—C (═O) —NH-alkyl group, 1 To three -NH-C (= O)- Alkyl group, one to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - alkyl group, one to three -S (= _{O) 2} —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 to 3 —NH ₂ group or 1 to 3 — (CH ₂ ) _0-3 optionally substituted by a Z group,
n is 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (— CH ₂ ) _0-3 Z ′, and Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NH— C (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) _0-3 Z ′,
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
“Alkyl” preferably represents C _1-10 -alkyl.

R ^8a preferably represents C _1-10 -alkyl.

HZ preferably represents a monocyclic or bicyclic heterocycle, which is a halogen, a C _1-10 -alkyl group, a C _6-10 -aryl group and a C _6-10 -aralkyl group (substituted by halogen). It may be substituted with one or more substituents selected from the group consisting of:

In the context of the present invention (ie in the above formula and also in the following formula) C _1-10 -alkyl represents a straight-chain or branched alkyl having 1 to 10 carbon atoms. Examples which may be mentioned as preferred are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl and tert-butyl.

In the context of the present invention C _{6-10 -aryl-} represents a monocyclic or bicyclic aromatic homocyclic ring, for example phenyl and naphthyl.

In the context of a C _6-10 -aralkyl basic invention, it represents a monocyclic aromatic homocyclic ring to which a C1-C4-alkyl group is attached, for example phenyl. An example of a C _6-10 -aralkyl group is benzyl.

C _5-10 -heteroaryl in the context of the present invention represents a monocyclic or bicyclic aromatic heterocycle having a total of 6 to 10 ring atoms, the rings being N, O, S, SO and SO _It contains 1 or 2 ring heteroatoms from the group consisting of ₂ and is bonded via a ring carbon atom or, optionally, a ring nitrogen atom. Examples that may be mentioned include pyridyl, furanyl, pyrimidyl, imidazolyl, thienyl, thiophenyl, isoxazoyl, isothiazoyl, 1,2,3-oxadiazoyl, furazanyl, 1,2,3-triazoyl (Triazoyl), 1,2,4-triazoyl (triazoyl), pyridazyl, pyrrolyl, triazinyl, indolyl, quinolinyl, quinazolinyl, 1,3-benzodioxole, isoindolyl, indazolyl, 1H-pyrazolo [3,4-d] Pyrimidyl, benzotriazolyl, isoquinolinyl, cinolinyl, phthalazinyl, pteridinyl, naphthyridinyl, benzoimidazolinyl, benzothiazolinyl, Zookisazoriniru is 3,4-methylenedioxyphenyl and benzo [6] furanyl.

Monocyclic or bicyclic heterocycle In the context of the present invention, it represents a monocyclic or bicyclic heterocycle having a total of 5 to 10 ring carbon atoms, the ring being N, O, S, SO and It contains 1 to 3 ring heteroatoms from the group consisting of SO ₂ and is bonded via a ring carbon atom or, where appropriate, a ring nitrogen atom. Examples that may be mentioned are piperidyl, pyrrolinyl, morpholinyl, 3,4-methylenedioxyphenyl and tetrahydrofuranyl.

  In the context of the present invention, a halogen atom represents F, Cl, Br or I.

  As used herein, the term “heteroalkyl” refers to one or more heteroatoms, ie, elements other than carbon that replace one or more carbon atoms (oxygen, sulfur, nitrogen, phosphorus, etc. (limited to these). A straight-chain or branched alkyl group including

  Whenever a group is described as being “substituted,” the group is substituted with one or more specified substituents. Where no substituent is indicated, it is indicated that the indicated “substituted” group is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl, heteroalicyclyl, aralkyl. , Heteroaralkyl, (heteroalicyclyl) alkyl, hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, carbamyl, thiocarbamyl, amide, sulfonamide, sulfonamide, carboxy, isocyanato, thiocyanato , Isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, Mino, which means that may be substituted with one or more groups selected mono-substituted amino group and a di-substituted amino groups, and from the protected derivatives thereof individually and independently.

  If the number of substituents is not specified (eg haloalkyl), one or more substituents may be present. For example, “haloalkyl” can include one or more of the same or different halogens. As another example, “C-C-alkoxyphenyl” can include one or more identical or different alkoxy groups containing one, two, or three atoms.

R ^1a , R ^2a , R ^3a , R ^4a , R at R ^1a , R ^2a , R ^4a or R ¹⁰ in substructure I (sub) or substructure II (sub) or at HZ in formula (Ia) ^By replacement of the hydrogen atom with ^8a or R ¹⁰ , the compound of formula (Ia) can be attached to the linker in a manner known to those skilled in the art. Particularly preferably, the hydrogen atom substitution takes place at R ^1a , R ^2a , R ^3a , R ^4a or at the pyrrolidine ring formed by R ^2a and R ^4a . This coupling can be performed chemically by various routes, as exemplarily shown in Schemes 2 to 16 in the Examples. In particular, it may be possible to modify a low molecular weight KSP inhibitor for attachment to a linker, for example by introduction of a protecting group or leaving group to facilitate substitution (in the reaction, a hydrogen atom Not said leaving group is replaced by a linker). Next, the KSP inhibitor-linker molecule thus obtained (the linker has a reactive group with respect to binding to the binder) is reacted with the binder to obtain the binder complex according to the present invention. it can. In the experimental part, this procedure is illustratively described by several examples.

Preferred for R ^1a are —H, —COOH, —C (═O) —NHNH ₂ , — (CH ₂ ) _1-3 NH ₂ , —C (═O) —NZ ″ (CH ₂ ) _1-3. a -NH ₂ and _{-C (= O) -NZ "CH} 2 COOH,
Z ″ represents —H or —NH ₂ .

Or Preferred for R ^2a and ^{R 4a} is ^{H, R 2a} and ^{R 4a} together form (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents -H.

Preferred for R ^3a is C _{1-10 -alkyl-} , which is —OH, —O-alkyl, —SH, —S-alkyl, —O—C (═O) -alkyl, —O—C (═ O) -NH-alkyl, -NH-C (= O) -alkyl, -NH-C (= O) -NH-alkyl, -S (= O) _n -alkyl, -S (= O) _2- NH Optionally substituted by -alkyl, -NH-alkyl, -N (alkyl) ₂ or -NH ₂ ,
Alkyl is preferably C _1-3 -alkyl,
n is 0, 1 or 2.

Preferred for R ^8a are branched C _1-5 -alkyl groups, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl and tert-butyl.

Preferred for HZ is a monocyclic or bicyclic heterocycle, which is a halogen, a C _1-10 -alkyl group, a C _6-10 -aryl group and a C _6-10 -aralkyl group (substituted by halogen). It may be substituted with one or more substituents selected from the group consisting of:

  Particularly preferably, HZ is a substituted pyrrole, pyrazole, imidazole, quinazoline or dihydroquinazoline, which is substituted in the ortho position (with respect to R1a etc. and the substituent) by an optionally substituted benzyl group. Furthermore, the substituted pyrrole, pyrazole, imidazole or quinazoline can preferably be substituted by oxo (in the case of dihydroquinazoline) or a phenyl group substituted by one or two halogen atoms. Particularly preferably, HZ is substituted pyrrole.

  A preferably used KSP inhibitor is ispinesive. A further preferred KSP inhibitor is Ary-520.

  Other particularly preferred compounds of structure KSP-L- are those having the following formula (IIa) or (II), and salts, solvates, solvate salts and epimers thereof:

Formula (IIa):

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or CF X ₂ represents C, X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH or CF, X ₂ represents N and X ₃ represents C;
(Preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N);
R ¹ is -H, -L- # 1, -MOD or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
R ² is -H, -L- # 1, -MOD, -C (= O) -CHY 4 -NHY 5 or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents —H, —L— # 1, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
Z is ^{-H, -OY} 3, -SY 3, represents -NHY 3, -C (= O) -NY 1 Y 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ⁴ represents R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) - or ^{_{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - group or a cathepsin cleavable group R _{^{21 - (C = O) (}} 0-1) - (P3) (1-2) -P2- represent,
R ²¹ is H, C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6- 10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - heterocyclic alkoxy group _(which, -NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (O) 2 -N ( alkyl _{2 -COOH, -C (= O) -NH} 2, -C (= O) -N ( _{alkyl) 2,} or may be substituted one or more times by -OH), -. H or a group - (O) _x - denotes the _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids; when there are a plurality of amino acids P3, P3 can have the same or different amino acids as defined above;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents —H, halogen (preferably —F), —NH ₂ , —SO ₃ H, —COOH, —SH or —OH, and the hydrogen atom of the secondary amino group in the pyrrolidine ring is R ²¹ —C ( _{= O) -P3 (0-2) -P2} -NH-CH (CH 2 C (= O) -NH 2) -C (= O) -SIG- it may be replaced by;
L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids; when there are a plurality of amino acids P3, P3 can have the same or different amino acids as defined above;
-SIG is a self-destructing group that gives a free secondary amine upon cleavage of the -C (= O) -SIG bond;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is -L- # 1, -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably C _1-10 -alkyl, C _6-10- Represents an aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, which is 1 to 3 —OH group, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 O-alkyl groups, 1 to 3 -SH group, 1 to 3 -S-alkyl group, 1 to 3 -O-C (= O) -alkyl group, 1 to 3 -O-C (= O) -NH-alkyl group 1 to 3 —NH C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, the one to three -S (= _{O) n} - alkyl group, one to three - S (═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 to 3 —NH ((CH ₂ CH ₂ O ) _1-20 H) group, optionally substituted by 1 to 3 —NH ₂ groups or 1 to 3 — (CH ₂ ) _0-3 Z groups,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
n represents 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NHC (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) _0-3 represents Z '
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
R ⁵ is —L— # 1, H, —MOD, —NH ₂ , —NO ₂ , halogen (especially —F, —Cl, —Br), —CN, —CF ₃ , —OCF ₃ , —CH _{2. F,} -CH 2 _F, -SH or represents - _{(CH 2) 0-3} Z,
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} - represents alkynyl, _hydroxy, -NO _2, -NH 2, -COOH, or halogen (especially -F, -Cl, -Br), and - alkynyl, fluoro _{-C 2-10}
R ⁸ is C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _2-10 -alkynyl, fluoro-C _2-10 -alkynyl , C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl, — or — (CH ₂ ) _0-2- (HZ ² ),
HZ ² represents a 4 to 7 membered heterocycle having 2 or less heteroatoms selected from the group consisting of N, O and S, each of these groups being —OH, —COOH or —NH ₂ or — Optionally substituted by L- # 1;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
One of the substituents R ¹ , R ² , R ³ , R ⁴ R ⁵ , R ⁸ and R ¹⁰ represents -L- # 1 (or in the case of R ⁸ ),
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ ;
In -MOD definition above _{^- (NR} 10) n ^- represents (G1) o -G2-G3,
R ¹⁰ represents —H or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is —NH—C (═O) — or

, R ¹⁰ does not represent —NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y —, —C (═O) —, —CR ^x = N—O— may be interrupted one or more times,
The hydrocarbon chain including any side chain is replaced by —NH—C (═O) —NH ₂ , —COOH, —OH, —NH ₂ , NH—CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid. May have been
R ^y represents —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NH—C (═O) —NH ₂ , — _{COOH, -OH, -NH 2, NH} -CN-NH 2, sulfonamide, sulfone, optionally substituted by a sulfoxide or sulfonic acid may,
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl;
G3 represents -H or -COOH;
-MOD preferably has at least one -COOH group.

Formula (II):

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or CF X ₂ represents C and X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH and X ₂ represents N, X ₃ represents C,

R ¹ is -H, -MOD, -L- # 1 or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ² is -L- # 1, H, -MOD, -C (= O) -CHY 4 -NHY 5 or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents —L— # 1, —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
Z is ^{-H, -OY} 3, -SY 3, represents -NHY 3, -C (= O) -NY 1 Y 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ⁴ represents R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) - or ^{_{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - group or a cathepsin cleavable group R _{^{21 - (C = O) (}} 0-1) - (P3) (1-2) -P2- represent,
R ²¹ is H, C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6- 10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl, - N _{(alkyl) 2, -NH-C (=} O) - alkyl, N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -S (=} O) 2 NH 2, -S (= O ) ₂ -N (A _{Kill) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _alkyl) may be substituted one or more times with ₂ or -OH, or -H or a group - ( _O) x _- (represents ^{_{CH 2 CH 2 O) y -R}} 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids; when there are a plurality of amino acids P3, P3 can have the same or different amino acids as defined above;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ is —H, halogen (preferably —F or —Cl), —NH ₂ , —SO ₃ H, —COOH, —SH, C _1-4 -alkyl, C _1-4 -haloalkyl, C _1-4. -Alkoxy, hydroxy-substituted C _1-4 -alkyl, —C (═O) —O— (C _1-4 -alkyl) or —OH, wherein the hydrogen atom of the secondary amino group in the pyrrolidine ring is R ^{_{21 -C (= O) -P3 (}} 0-2) -P2-NH-CH (CH 2 C (= O) -NH 2) -C (= O) -SIG- may be replaced by;
L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
-SIG is a self-destructing group that gives a free secondary amine upon cleavage of the -C (= O) -SIG bond;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is -L- # 1, -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably -L- # 1 or C _1-10 -alkyl , C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl Group, 1 to 3 —SH group, 1 to 3 —S-alkyl group, 1 to 3 —O—C (═O) -alkyl group, 1 to 3 —O—C (═ O) -NH-alkyl group, Of three -NH-C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - group 1 to 3 —S (═O) ₂ —NH-alkyl groups, 1 to 3 —NH-alkyl groups, 1 to 3 —N (alkyl) ₂ groups, 1 to 3 —NH _Optionally substituted by ₂ or 1 to 3 — (CH ₂ ) _0-3 Z groups,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
n represents 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NHC (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) _0-3 represents Z '
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁵ is _-H, -NH _2, -NO _2, halogen (especially -F, -Cl, -Br), - SH or - _{(CH 2)} represents a _0-3 Z,
Z represents -H, -OY ³ , -SY ³ , halogen, -NHY ³ , -C (O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} -alkynyl, fluoro- _C2-10 -alkynyl, hydroxy or halogen (especially -F, -Cl, -Br);
R ⁸ represents C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl, or optionally substituted oxetane;
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ ;
And salts, solvates, solvate salts and epimers thereof.

Replacement of a hydrogen atom with R ¹ , R ² , R ³ , R ⁴ , R ⁵ or R ⁸ in the form known to those skilled in the art or with the pyrrolidine ring (R ¹⁰ ) formed by R ² and R ⁴ A linker of the compound of formula (IIa) or (II) in which none of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents -L- # 1 Can be combined. Thereby, one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ or R ¹⁰ represents -L- # 1, L represents a linker, and # 1 represents a binder or A complex of formula (IIa) or (II) is obtained which represents the binding of the derivative of Thus, when the KSP inhibitor according to formula (IIa) or (II) is bound to a binder, ^{one of} the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ or R ¹⁰ is -L- # 1, L represents a linker, and # 1 represents a bond to a binder or a derivative thereof. That is, in the case of the complex, one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents -L- # 1, and -L- # 1 represents a binder. For example, binding to an antibody. Particularly preferably, one of the substituents R ¹ and R ³ represents -L- # 1. In this embodiment, ^{R 4} is as defined above _{^{-H, R 21 - (C =}} O) (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 C (= _{O) -NH 2) -C (=} O) - or _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C ( ═O) — is preferred. In another preferred embodiment, the substituent R ⁴ represents -L- # 1, and the linker is cleavable at the nitrogen atom bonded to R ⁴ so that cleavage results in a primary amino group (R ⁴ = Equivalent to -H). The corresponding cleavable group will be described below.

When R ¹ is not —H, the carbon atom bonded to R ¹ is a stereogenic center, which can exist in the L and / or D configuration, preferably in the L configuration.

When R ² is not, the carbon atom bonded to R ² is a stereogenic center and it can exist in the L and / or D configuration.

  The binder is preferably a human, humanized or chimeric monoclonal antibody or antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or antigen-binding fragment thereof or an anti-EGFR antibody or antigen-binding fragment thereof. Particularly preferred is an anti-TWEAKR antibody that specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169), particularly anti-TWEAKR antibody TPP-2090, or anti-Her2 antibody trastuzumab. All of the described antibodies include deglycosylated variants of these antibodies caused by deglycosylation with PNGaseF or by mutation of either heavy chain N297 (Kabat numbering) to any amino acid.

One of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents -L- # 1;
X ₁ represents N, X ₂ represents N, X ₃ represents C;
X ₁ represents CH or CF, X ₂ represents C, and X ₃ represents N;
Formula (IIa) or (II) wherein X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH, X ₂ represents N, and X ₃ represents C A compound of
Particularly, _{X 1} represents N, _{X 2} represents N, _{X 3} represents C; or _{X 1} represents CH, _{X 2} represents a C, _{which X 3} represents N are particularly preferred. Particularly preferred are compounds in which X ₁ represents CH, X ₂ represents C, and X ₃ represents N.

  Regarding A, preferred is -C (= O)-.

Preferred regard R ¹ _{is, -L- # 1, -H, -COOH} , -C (= O) -NHNH 2, - (CH 2) 1-3 NH 2, -C (= O) -NZ "( CH ₂ ) _1-3 NH ₂ and —C (═O) —NZ ″ CH ₂ COOH, wherein Z ″ represents —H or —NH ₂ .

R ² and R ⁴ represent —H, or R ² represents —H, and R ⁴ represents R ²¹ — (C═O) ( _0-1) — (P3) _(0-2) —P2-NH _{-CH (CH 2 C (= O} ) -NH 2) -C (= O) - and represents, or ^{R 2} and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -is represented, and R ¹⁰ represents -H or -L- # 1.

Preferred for R ³ is —L— # 1 or C _{1-10 -alkyl-} , which is —OH, —O-alkyl, —SH, —S-alkyl, —O—C (═O) -alkyl. , -O-C (= O) -NH-alkyl, -NH-C (= O) -alkyl, -NH-C (= O) -NH-alkyl, -S (O) _n -alkyl, -S ( ═O) ₂ —NH-alkyl, optionally substituted by —NH-alkyl, —N (alkyl) ₂ or —NH ₂ ; alkyl is preferably C _1-3 -alkyl, n is 0, 1 Or 2.

Preferred for R ⁵ is -L- # 1, -H or -F.

Preferred for R ⁶ and R ⁷ are, independently of one another, —H, (optionally fluorinated) C _1-3 -alkyl, (optionally fluorinated) C _2-4 -alkenyl, ( _C2-4 -alkynyl, hydroxy or halogen, which may be fluorinated.

Preferred for R ⁸ is a branched C _1-5 -alkyl group, particularly a group of formula —C (CH ₃ ) ₂ — (CH ₂ ) _0-2- R ^y , where R ^y is —H, — OH, -COOH, it represents a -NH ₂ or-L-# 1. Particularly preferred are the formula _{-C (CH 3) 2 - (} CH 2) a group of -R ^{y, R y} represents -H or-L-# 1.

Preferred for R ⁹ is -H or -F.

Particularly preferred is
None or ^{one of} the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents -L- # 1;
X ₁ represents N, X ₂ represents N, X ₃ represents C;
X ₁ represents CH or CF, X ₂ represents C, and X ₃ represents N;
X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH, X ₂ represents N, X ₃ represents C, A represents —C (═O) -Represents;
R ¹ is —H, —COOH, —C (═O) —NHNH ₂ , — (CH ₂ ) _1-3 NH ₂ , —C (═O) —NZ ″ (CH ₂ ) _1-3 —NH ₂ and -C (= O) _-NZ "represents a CH 2 COOH, Z" represents -H or -NH _2;
R ² and ^{R 4} represents represents or ^{R 2} is -H and -H, ^{R 4} is _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH _{(CH 2 C (= O)} -NH 2) -C (= O) - (. P2 and P3 have the same meaning as defined above) represents, or ^{R 2} and ^{R 4} together (pyrrolidine forming a _ring), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - ^{represents, R 10} represents H or-L-# 1;
R ³ represents a phenyl group which may be mono- or poly-substituted by halogen (particularly F) or C _1-3 -alkyl which may be fluorinated, or C ₁ which may be fluorinated. _-10 -alkyl group [it is -OY ⁴ , -SY ⁴ , -O-C (= O) -Y ⁴ , -O-C (= O) -NH-Y ⁴ , -NH-C (= O) ^{-Y 4, -NH-C (=} O) -NH-Y 4, -S (= O) n -Y 4 (n represents 0, 1 or _{2.), - S (=} O) 2 -NH -Y ⁴ , —NH—Y ⁴ or —N (Y ⁴ ) ₂ may be substituted, and Y ⁴ may be —H, phenyl (halogen (particularly F), or fluorinated C _{1- 3} - alkyl by may be monosubstituted or polysubstituted) or alkyl (alkyl group, -O , -COOH and / or -NHC (= _{O) -C 1-3} - may be substituted by alkyl, the alkyl is _{preferably, C 1-3} - it represents a) alkyl.. ];
Particularly preferably, R ³ is —OH, —O-alkyl, —SH, —S-alkyl, —O—C (═O) -alkyl, —O—C (═O) —NH-alkyl, —NH—. C (= O) -alkyl, -NH-C (= O) -NH-alkyl, -S (= O) _n -alkyl (n represents 0, 1 or 2), -S (= O) _2. Optionally substituted by -NH-alkyl, -NH-alkyl, -N (alkyl) ₂ or -NH ₂ (wherein alkyl preferably denotes C _1-3 -alkyl);
R ⁵ represents -H or -F;
R ⁶ and R ⁷ are independently of each other —H, (optionally fluorinated) C _1-3 -alkyl, (optionally fluorinated) C _2-4 -alkenyl, (fluorinated) May represent) C _2-4 -alkynyl, hydroxy or halogen;
R ⁸ represents a branched C _1-5 -alkyl group;
Compounds of formula (IIa) or (II), wherein R ⁹ represents —H or —F.

In addition (alone or in combination),
R ¹ represents -L- # 1, -COOH or -H,
● ^{R 2} and ^{R 4} represent independently of one another to-L-# 1 or -H; or ^{R 2} represents -H, ^{R 4} is _{^{R 21 - (C = O)}} (0-1) - (P3) _{(0-2) -P2-NH-CH} (CH 2 C (= O) -NH 2) -C (= O) - (P2 and P3 have the same meanings as defined above.) represent, or ^{R 2} and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - ^{represents, R 10} represents -H or-L-# 1,
● A represents -C (= O)-
● ^{R 3} is _{- (CH 2) OH, -CH} (CH 3) OH, -CH 2 -S-CH 2 CH (COOH) NH-C (= O) -CH 3, -CH (CH 3) OCH 3 Represents a phenyl group (which may be substituted by 1 to 3 halogen atoms, 1 to 3 amino groups or 1 to 3 alkyl groups which may be halogenated), or- L- # 1
● R ⁵ represents -L- # 1 or -H,
R ⁶ and R ⁷ independently of one another represent —H, C _1-3 -alkyl or halogen; in particular, R ⁶ and R ⁷ represent —F;
• R ⁸ represents C _1-4 -alkyl (preferably tert-butyl); and / or • R ⁹ represents —H;
Preferred is when one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents -L- # 1.

Furthermore, according to the present invention,
R ¹ represents -L- # 1, -COOH or -H,
● ^{R 2} and ^{R 4} represent independently of one another to-L-# 1 or -H, or ^{R 2} represents -H, ^{R 4} is _{^{R 21 - (C = O)}} (0-1) - (P3) _{(0-2) -P2-NH-CH} (CH 2 C (= O) -NH 2) -C (= O) - (P2 and P3 have the same meanings as defined above.) represent, or ^{R 2} and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - ^{represents, R 10} represents -H or-L-# 1,
● A represents -C (= O)-
● ^{R 3} is _{- (CH 2) OH, -CH} (CH 3) OH, -CH 2 -S-CH 2 CH (COOH) NH-C (= O) -CH 3, -CH (CH 3) OCH 3 A phenyl group [optionally substituted by 1 to 3 halogen atoms, 1 to 3 amino groups or 1 to 3 alkyl groups (which may be halogenated). Represents -L- # 1,
● R ⁵ represents -L- # 1 or -H,
R ⁶ and R ⁷ independently of one another represent —H, C _1-3 -alkyl or halogen; in particular, R ⁶ and R ⁷ represent —F;
● R ⁸ represents C _1-4 -alkyl (preferably tert-butyl);
● R ⁹ represents -H,
Preferred is when one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents -L- # 1.

  Other particularly preferred compounds are those having the following formula (IIIa) or (III), and salts, solvates and solvate salts thereof:

Formula (IIIa):

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or CF X ₂ represents C and X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH and X ₂ represents N, X ₃ represents C,
(Preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N);
R ¹ represents —H, —L-BINDER, —MOD or — (CH ₂ ) _0-3 Z;
Z is ^{-H, -NHY 3, -OY 3,} -SY 3, represents halogen, ^{-C (= O) -NY 1 Y} 2 or -C (= O) -OY ^3,
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ² represents -L-BINDER, -H, -MOD, -C (= O) -CHY ⁴ -NHY ⁵ or-(CH ₂ ) _0-3 Z;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents -L-BINDER, -H, -C (O) -CHY ⁴ -NHY ⁵ or-(CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ⁴ represents R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) -Represents a group of
R ²¹ represents —H, C _1-10 -alkyl-, C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6. -10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy - , _{C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (= O) 2 -N (Al _{Le) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _{alkyl) 2} or may optionally be substituted one or more times by -OH,, or -H or a group - a _{(CH 2 CH 2 O) y} -R 22, - O x
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably C1-12- _alkyl), - _CH 2 -COOH, represents _-CH 2 _-CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents _{-L-BINDER, -H, -NH 2} , -SO 3 H, -COOH, a -SH or -OH;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is —L-BINDER, —MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably —L-BINDER, or C _1-10 -alkyl, _{Represents a} C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, It has 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl groups 1 to 3 —SH groups, 1 to 3 —S-alkyl groups, 1 to 3 —O—C (═O) -alkyl groups, 1 to 3 —O—C (═O ) − H-alkyl group, 1 to 3 —NH—C (═O) -alkyl group, 1 to 3 —NH—C (═O) —NH-alkyl group, 1 to 3 —S (═ O) _n -alkyl group, 1 to 3 —S (═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 May be substituted by 1 to 3 —NH ₂ groups or 1 to 3 — (CH ₂ ) _0-3 Z groups,
n is 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ is _{-H, - (CH 2) 0-3} -CH (NH-C (= O) -CH 3) Z ', - (CH 2) 0-3 -CH (NH 2) Z' or - ( CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
“Alkyl” preferably represents C _1-10 -alkyl);
R ⁵ is -L-BINDER, -H, -NH ₂ , -NO ₂ , halogen (especially F, Cl, Br), -CN, -CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F , -SH or - it represents _{(CH 2) 0-3} Z,
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} - represents alkynyl, _hydroxy, -NO _2, -NH 2, -COOH, or halogen (especially -F, -Cl, -Br), and - alkynyl, fluoro _{-C 2-10}
R ⁸ is C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl,
C _2-10 -alkynyl, fluoro-C _2-10 -alkynyl, C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl or — (CH ₂ ) _0-2- (HZ ² )
HZ ² represents a 4 to 7 membered heterocyclic ring (preferably oxetane) having 2 or less heteroatoms selected from the group consisting of N, O and S, each of these groups being —OH, —COOH or It may be substituted by -NH ₂ or -L-BINDER;
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ ;
L represents a linker;
BINDER represents a binder or a derivative thereof, and the binder may be bound to a plurality of active compound molecules,
One representative of R ¹ , R ² , R ³ R ⁴ , R ⁵ and R ⁸ represents -L-binder;
-MOD represents-(NR _< ¹⁰ _> ) _n- (G1) _o -G2-H,
R ¹⁰ represents —H or C ₁ -C ₃ -alkyl;
G1 represents —NHC (═O) —, —C (═O) —NH—, or

(G1 is —NH—C (═O) — or

R ¹⁰ does not represent NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y — may be interrupted one or more times by one or more of:
R ^y represents —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NH—C (═O) —NH ₂ , — _{COOH, -OH, -NH 2, NH} -CN-NH 2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid, or ^{-C (= O) -, -} CR x = N-O- Represents
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl;
Hydrocarbon chain containing either side _{chains, -NH-C (= O)} -NH 2, -COOH, -OH, -NH 2, NH-CN-NH 2, sulfonamide, sulfone, sulfoxide or sulfone acid May be replaced by
-MOD preferably has at least one group -COOH.

In the case of the binder complex of the KSP inhibitor of formula (IIIa), at most one representative of R ¹ , R ² , R ³ R ⁴ , R ⁵ , R ⁸ and R ¹⁰ (in the conditions provided above Can represent -L-BINDER, L represents a linker, BINDER represents a binder or a derivative thereof, and the binder may be bound to a plurality of active compound molecules. good.

Formula (III):

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C, or X ₁ represents CH, X ₂ represents C, X ₃ represents N, or X ₁ represents NH X ₂ represents C, X ₃ represents C, or X ₁ represents CH, X ₂ represents N, and X ₃ represents C;
R ¹ represents -H, -L-BINDER or-(CH ₂ ) _0-3 Z;
Z is ^{-H, -NHY 3, -OY 3,} -SY 3, represents halogen, ^{-C (= O) -NY 1 Y} 2 or -C (= O) -OY ^3,
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ² and R ⁴ each independently represent -L-BINDER, -H, -C (O) -CHY ⁴ -NHY ⁵ or-(CH ₂ ) _0-3 Z;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ is _L-# 1, represents _{-H, -NH 2, -SO 3 H} , -COOH, -SH, or -OH;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is —L-BINDER or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably —L-BINDER, or C _1-10 -alkyl, C _{6− Represents a 10} -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl groups, 1 to 3 —SH groups, 1 to 3 —S-alkyl groups, 1 to 3 —O—C (═O) -alkyl groups, 1 to 3 —O—C (═O) —NH -Al Group, one to three -NH-C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= O) _n -alkyl group, 1 to 3 —S (═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 to 3 Optionally substituted by 1 —NH ₂ group or 1 to 3 — (CH ₂ ) _0-3 Z groups,
n is 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ is _{-H, - (CH 2) 0-3} -CH (NH-C (= O) -CH 3) Z ', - (CH 2) 0-3 -CH (NH 2) Z' or - ( CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
“Alkyl” preferably represents C _1-10 -alkyl);
R ⁵ is -L-BINDER, -H, -NH ₂ , -NO ₂ , halogen (especially F, Cl, Br), -CN, -CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F , -SH or - it represents _{(CH 2) 0-3} Z,
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} -alkynyl, fluoro- _C2-10 -alkynyl, hydroxy, or halogen (especially -F, -Cl, -Br);
R ⁸ represents C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl or optionally substituted oxetane;
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ ;
-L represents a linker;
-Binder represents a binder or a derivative thereof, and the binder may be bound to a plurality of active compound molecules;
One representative of R ¹ , R ² , R ³ R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents -L-binder;
And salts, solvates and solvate salts thereof.

  Further preferred according to the present invention is a complex of the following KSP inhibitors:

Formula (IIIb):

Where
X ₁ , X ₂ and X ₃ have the same meaning as in formula (IIIa) or (III) (preferably X ₁ represents —CH—, X ₂ represents —C—, and X ₃ represents -N-)), R ¹ , R ² , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the same meaning as in formula (IIIa) or (III), A represents —C (═O) —, B represents a single bond, —O—CH ₂ — or —CH ₂ —O—, and R ²⁰ represents —NH ₂ , —F, —CF ₃ or —CH _3. N represents 0, 1 or 2.

Formula (IIIc):

Where
X ₁ , X ₂ , X ₃ are in formula (IIIa) or (III) (preferably X ₁ represents —CH—, X ₂ represents —C—, and X ₃ represents —N—). Has the same meaning as
A, R ¹ , R ³ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the same meaning as in formula (IIIa) or (III), and A is preferably —C (═O) —. R ³ represents —CH ₂ OH, —CH ₂ OCH ₃ , —CH (CH ₃ ) OH or —CH (CH ₃ ) OCH ₃ .

Formula (IIId):

Where
X ₁ , X ₂ and X ₃ have the same meaning as in formula (IIIa) or (III) (preferably X ₁ represents —CH—, X ₂ represents —C—, and X ₃ represents Represents -N-);
A, R ³ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the same meaning as in formula (IIIa) or (III), A preferably represents —C (═O) — R ³ represents —CH ₂ —S _x — (CH ₂ ) _0-4 —CHY ⁵ —COOH, x is 0 or 1, Y ⁵ represents —H or —NHY ⁶ , and Y ⁶ represents —H. or an -C (= O) -CH _3.

Formula (IIIe):

Where
X ₁ represents —CH—, X ₂ represents —C—, and X ₃ represents —N—;
A, R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the same meaning as in formula (IIIa) or (III), and R ¹ represents —L-BINDER.

Furthermore, in the compounds of formula (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe) (alone or in combination)
● Z represents Cl or Br;
R ¹ represents — (CH ₂ ) _0-3 Z, Z represents —C (═O) —NY ¹ Y ² , and Y ² represents — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ and Y ¹ represents —H, —NH ₂ or — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′;
Y ¹ represents —H, Y ² represents — (CH ₂ CH ₂ O) ₃ —CH ₂ CH ₂ Z ′, Z ′ represents —COOH;
Y ¹ represents —H, Y ² represents —CH ₂ CH ₂ Z ′, and Z ′ represents — (C (═O) —NHCHY ⁴ ) ₂ COOH;
Y ¹ represents —H, Y ² represents —CH ₂ CH ₂ Z ′, Z ′ represents — (C (═O) —NHCHY ⁴ ) ₂ COOH, and one of the Y ⁴ groups is i-propyl. And the other represents — (CH ₂ ) ₃ —NH—C (═O) —NH ₂ ;
Y ¹ represents H, Y ² represents —CH ₂ CH ₂ Z ′, Z ′ represents — (C (═O) —NHCHY ⁴ ) ₂ COOH, and one of the Y ⁴ groups represents —CH ₃ . The other represents — (CH ₂ ) ₃ —NH—C (═O) —NH ₂ ;
Y ⁴ represents a linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ ;
At least one representative of Y ⁴ is selected from the group consisting of i-propyl and —CH ₃ ;
Y ¹ represents —H, Y ² represents —CH ₂ CH ₂ Z ′, Z ′ represents —C (═O) —NHCHY ⁴ COOH, and Y ⁴ may be substituted by —NH ₂ Represents good aryl or benzyl;
● Y ⁴ represents aminobenzyl;
● ^{R 2} is - _{(CH 2) 0-3} represents Z, Z represents -SY ^3;
R ⁴ represents —C (═O) —CHY ⁴ —NHY ⁵ and Y ⁵ represents —H;
R ⁴ represents —C (═O) —CHY ⁴ —NHY ⁵ and Y ⁵ represents —C (═O) —CHY ⁶ —NH ₂ ;
R ⁴ represents R ²¹ -L-Ala-L-Ala-L-Asn- #;
● It is preferable that Y ⁴ represents a linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ .

Further, when R ¹ , R ² or R ³ in formula (IIa) or (IIIa) represents -MOD, particularly when R ⁴ represents -L- # 1 or -L-BINDER (especially -L is , -N-R ⁴ or -N-L- # 1 or -L-BINDER, where R ⁴ or L is replaced by -H.

Particularly preferably, R ³ represents -MOD, R ¹ or R ⁴ represents -L- # 1 or -L-BINDER,
-MOD represents-(NR _< ¹⁰ _> ) _n- (G1) _o -G2-H,
R ¹⁰ represents —H or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is —NH—C (═O) — or

R ¹⁰ does not represent NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y — may be interrupted one or more times by one or more of:
R ^y represents —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NH—C (═O) —NH ₂ , — _{COOH, -OH, -NH 2, NH} -CN-NH 2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid, or ^{-C (= O) -, -} CR x = N-O- Represents
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl;
Hydrocarbon chain containing either side _{chains, -NH-C (= O)} -NH 2, -COOH, -OH, -NH 2, -NH-CN-NH 2, sulfonamide, sulfone, sulfoxide, or sulfone May be substituted by an acid,
-MOD preferably has at least one group -COOH.

Particularly preferably, the group -MOD has a (preferably terminal) -COOH group, for example a betaine group. Preferably, groups -MOD has the formula _-CH 2 _-S x _- has a ^{_{(CH 2) 0-4 -CHY 5 -COOH}} , x is 0 or 1, ^{Y 5} is -H or -NHY ⁶ Y ⁶ represents —H or —C (═O) —CH ₃ .

Other particularly preferred compounds are those having the following formula (IV), and salts, solvates, solvate salts and epimers thereof:

Where
X ₁ represents N, X ₂ represents C, X ₃ represents N;
R ¹ represents -H, -L-BINDER or-(CH ₂ ) _0-3 Z;
Z is ^{-H, -NHY 3, -OY 3,} -SY 3, represents halogen, ^{-C (= O) -NY 1 Y} 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ or —CH (CH ₂ W) Z ′. ,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ² and R ⁴ each independently represent —L-BINDER, —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ is _L-# 1, represents _{-H, -NH 2, -SO 3 H} , -COOH, -SH, or -OH;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is an optionally -L-BINDER or substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, or _{-CH 2 -S x, - (CH} 2) 0-4 -CHY 5 Represents -COOH,
x is 0 or 1,
Y ⁵ represents -H or -NHY ⁶ ;
Y ⁶ represents —H or —C (═O) —CH ₃ ,
Preferably -L-BINDER or _{C 1-10,} - _{alkyl, C 6-10} - aryl or _{C 6-10} - _{aralkyl, C 5-10} - _{heteroalkyl, C 1-10} - alkyl _{-O-C 6-10 -Represents an} aryl or C _{5-10 -heterocyclic} alkyl group, which has 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (1 to 3 halogen atoms each) 1 to 3 —O-alkyl groups, 1 to 3 —SH groups, 1 to 3 —S-alkyl groups, 1 to 3 —O—C (═O) — An alkyl group, 1 to 3 —O—C (═O) —NH-alkyl group, 1 to 3 —NH—C (═O) -alkyl group, 1 to 3 —NH—C (═ O) -NH-alkyl group, 1 to 3 -S (= O) _n -alkyl 1 to 3 —S (═O) ₂ —NH-alkyl groups, 1 to 3 —NH-alkyl groups, 1 to 3 —N (alkyl) ₂ groups, 1 to 3 Optionally substituted by —NH ₂ groups or 1 to 3 — (CH ₂ ) _0-3 Z groups,
n is 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ is _{-H, - (CH 2) 0-3} -CH (NH-C (= O) -CH 3) Z ', - (CH 2) 0-3 -CH (NH 2) Z' or - ( CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
“Alkyl” preferably represents C _1-10 -alkyl);
R ⁵ represents -L-BINDER, -H, -NH ₂ , -NO ₂ , halogen (especially F, Cl, Br), -SH or-(CH ₂ ) _0-3 Z;
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
L represents a linker;
BINDER represents a binder or derivative thereof, which may be bound to a plurality of active compound molecules;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _2−. Represents ₁₀ -alkynyl, fluoro- _C2-10 -alkynyl, hydroxy or halogen (especially -F, -Cl, -Br);
R ⁸ represents C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl or optionally substituted oxetane;
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ ;
^However, R 1, ^{R 2} and ^{R 4} are not to represent -H simultaneously.

Furthermore, in the compounds of formula (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) and (IV) (alone or in combination)
● Z represents Cl or Br;
R ¹ represents — (CH ₂ ) _0-3 Z, Z represents —C (═O) —NY ¹ Y ² , and Y ² represents — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ and Y ¹ represents —H, —NH ₂ or — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′;
Y ¹ represents —H, Y ² represents — (CH ₂ CH ₂ O) ₃ —CH ₂ CH ₂ Z ′, Z ′ represents —COOH;
Y ¹ represents —H, Y ² represents —CH ₂ CH ₂ Z ′, and Z ′ represents — (C (═O) —NHCHY ⁴ ) ₂ COOH;
Y ¹ represents —H, Y ² represents —CH ₂ CH ₂ Z ′, Z ′ represents — (C (═O) —NHCHY ⁴ ) ₂ COOH, and one of Y ⁴ representatives Represents i-propyl and the other represents — (CH ₂ ) ₃ —NH—C (═O) —NH ₂ ;
Y ¹ represents H, Y ² represents —CH ₂ CH ₂ Z ′, Z ′ represents — (C (═O) —NHCHY ⁴ ) ₂ COOH, and one representative of Y ⁴ is Represents —CH ₃ and the other represents — (CH ₂ ) ₃ —NH—C (═O) —NH ₂ ;
Y ⁴ represents a linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ ;
At least one representative of Y ⁴ is selected from the group consisting of i-propyl and —CH ₃ ;
Y ¹ represents —H, Y ² represents —CH ₂ CH ₂ Z ′, Z ′ represents —C (═O) —NHCHY ⁴ COOH, and Y ⁴ may be substituted by —NH ₂ Represents good aryl or benzyl;
● Y ⁴ represents aminobenzyl;
● ^{R 2} is - _{(CH 2) 0-3} represents Z, Z represents -SY ^3;
R ⁴ represents —C (═O) —CHY ⁴ —NHY ⁵ and Y ⁵ represents —H;
R ⁴ represents —C (═O) —CHY ⁴ —NHY ⁵ and Y ⁵ represents —C (═O) —CHY ⁶ —NH ₂ ;
R ⁴ represents R ²¹ -L-Ala-L-Ala-L-Asn- #;
● It is preferable that Y ⁴ represents a linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ .

The preferred ones are
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or CF X ₂ represents C, X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH or CF, X ₂ represents N and X ₃ represents C;
(Preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N);
R ¹ is H, -L- # 1 or -L-BINDER, -MOD or represents - _{(CH 2) 0-3} Z,
Z is ^{-H, -NHY 3, -OY 3,} -SY 3, represents halogen, ^{-C (= O) -NY 1 Y} 2 or -C (= O) -OY ^3,
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′ ) Or —CH (CH ₂ W) Z ′,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents —H or —OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ Representation;
R ² represents —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² independently of each other represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ Represent,
Y ⁵ is -H or -C (= O) represents the -CHY ⁶ -NH _2,
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents -H or R ²¹ -L-Ala-L-Ala-L-Asn-;
R ²¹ is —H, C _1-10 -alkyl-, C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6. -10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy - , _{C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (= O) 2 -N (Al _{Le) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _{alkyl) 2} or may optionally be substituted one or more times by -OH,, or -H or a group - a _{(CH 2 CH 2 O) y} -R 22, - O x
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2 represents;
A is -C (= O) -, - S (= O) -, - S (= O) 2 -, - S (= O) 2 -NH- or _{-C (= N-NH 2)} - a represents ;
R ³ is -L- # 1 or -L-BINDER, -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably -L-BINDER, or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -hetero Represents a ring alkyl group, which has 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 -O-alkyl group, 1 to 3 -SH group, 1 to 3 -S-alkyl group, 1 to 3 -O-C (= O) -alkyl group, 1 to 3- -C (= O) -NH-alkyl group, 1 to 3 -NH-C (= O) -alkyl group, 1 to 3 -NH-C (= O) -NH-alkyl group, 1 to 3 —S (═O) _n -alkyl groups, 1 to 3 —S (═O) ₂ —NH-alkyl groups, 1 to 3 —NH-alkyl groups, 1 to 3 —N (Alkyl) ₂ groups, 1 to 3 —NH ₂ groups or 1 to 3 — (CH ₂ ) _0-3 Z groups may be substituted,
n is 0, 1 or 2;
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (O) —OY ³ ;
Y ¹ and Y ² independently of each other represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ is —H, — (CH ₂ ) _0-3 —CH (NH—C (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — ( CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
“Alkyl” preferably represents C _1-10 -alkyl);
R ⁵ is —H, —MOD, —NH ₂ , —NO ₂ , halogen (particularly F, Cl, Br), —CN, —CF ₃ , —OCF ₃ , —CH ₂ F, —CH ₂ F, — Represents SH or — (CH ₂ ) _0-3 Z;
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² independently of each other represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} - represents alkynyl, _hydroxy, -NO _2, -NH 2, -COOH, or halogen (especially -F, -Cl, -Br), and - alkynyl, fluoro _{-C 2-10}
R ⁸ is C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _2-10 -alkynyl, fluoro-C _2-10 -alkynyl , C _4-10 -cycloalkyl or fluoro-C _4-10 -cycloalkyl,
One of the substituents R ¹ and R ³ represents -L- # 1 or -L-BINDER;
L represents a linker;
# 1 represents binding to the binder or derivative thereof;
Binder represents the binder,
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ ;
-MOD is _{^- (NR} 10) n ^- represents (G1) o -G2-G3,
R ¹⁰ represents —H or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH— or

(G1 is —NH—C (═O) — or

, R ¹⁰ does not represent —NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and has the groups —O—, —S—, —S (═O) —, —S (═O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y — may be interrupted one or more times by one or more of:
R ^y represents —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NH—C (═O) —NH ₂ , — _{COOH, -OH, -NH 2, NH} -CN-NH 2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid, or ^{-C (= O) -, -} CR x = N-O- Represents
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl,
The hydrocarbon chain including any side chain is —NH—C (═O) —NH ₂ , —COOH, —OH, —NH ₂ , —NH—CN—NH ₂ , sulfonamide, sulfone, sulfoxide or sulfone. May be substituted by an acid,
G3 represents -H or -COOH;
-MOD preferably has at least one group -COOH, of formula (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IV And the salts, solvates, solvate salts and epimers thereof.

Preferred is further
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or CF X ₂ represents C, X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH or CF, X ₂ represents N and X ₃ represents C;
(Preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N);
R ¹ is -H, -L- # 1 or -L-BINDER, -MOD or represents - _{(CH 2) 0-3} Z,
Z is ^{-H, -NHY 3, -OY 3,} -SY 3, represents halogen, ^{-C (= O) -NY 1 Y} 2 or -C (= O) -OY ^3,
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′ ) Or —CH (CH ₂ W) Z ′,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents —H or —OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ Representation;
R ² represents —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² independently of each other represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ Represent,
Y ⁵ is -H or -C (= O) represents the -CHY ⁶ -NH _2,
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents -H or R ²¹ -L-Ala-L-Ala-L-Asn-;
R ²¹ is —H, C _1-10 -alkyl-, C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6. -10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy - , _{C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (= O) 2 -N (Al _{Le) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _{alkyl) 2} or may optionally be substituted one or more times by -OH,, or -H or a group - a _{(CH 2 CH 2 O) y} -R 22, - O x
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2 represents;
A is -C (= O) -, - S (= O) -, - S (= O) 2 -, - S (= O) 2 -NH- or _{-C (= N-NH 2)} - a represents ;
R ³ is -L- # 1 or -L-BINDER, -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably C _1-10 -alkyl, _{Represents a} C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, It has 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl groups 1 to 3 —SH groups, 1 to 3 —S-alkyl groups, 1 to 3 —O—C (═O) -alkyl groups, 1 to 3 —O—C (═O ) -NH-Al Group, one to three -NH-C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= O) _n -alkyl group, 1 to 3 —S (═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 to 3 Optionally substituted by 1 —NH ₂ group or 1 to 3 — (CH ₂ ) _0-3 Z groups,
n is 0, 1 or 2;
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (O) —OY ³ ;
Y ¹ and Y ² independently of each other represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ is —H, — (CH ₂ ) _0-3 —CH (NH—C (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — ( CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
“Alkyl” preferably represents C _1-10 -alkyl);
R ⁵ is _{-MOD, -H, -NH 2, -NO} 2, halogen (particularly _{F, Cl, Br), -} CN, -CF 3, -OCF 3, -CH 2 F, -CH 2 F, - Represents SH or — (CH ₂ ) _0-3 Z;
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² independently of each other represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ independently of each other represent —H or halogen (particularly —F, —Cl, —Br),
R ⁸ represents C _1-10 -alkyl or fluoro-C _1-10 -alkyl,
One of the substituents R ¹ and R ³ represents -L- # 1 or -L-BINDER;
L represents a linker;
# 1 represents binding to the binder or derivative thereof;
Binder represents the binder,
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ ;
-MOD is _-CH 2 _-S x _- represents a ^{_{(CH 2) 0-4 -CHY 5 -COOH}} ,
x is 0 or 1,
Y ⁵ represents H or NHY ⁶ ;
Y ⁶ represents H or —C (═O) —CH ₃ , formula (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or Compounds of (IV), and salts, solvates, solvate salts and epimers thereof.

Particularly preferred according to the invention are compounds of the following formulas V, VI and VII (R ¹ , R ² , R ³ , R ⁴ and R ⁵ have the meanings mentioned above (for example of formula (IIa) or (IIIa As mentioned for)))).

Particularly preferred are R ¹ and R ⁵ represent -H or -L- # 1; R ² and R ⁴ independently of each other represent -L- # 1 or -H, or R ² and R ⁴ together (Forms a pyrrolidine ring), —CH ₂ —CHR ¹⁰ — or —CHR ¹⁰ —CH ₂ —, R ¹⁰ represents —H or —L— # 1; R ³ represents —CH ₂ OH, —CH (CH ₃ ) OH or —L— # 1, wherein one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents —L— # 1 It is a compound of VII. Particularly preferred are the corresponding compounds of formula VI.

Preferred antibody drug conjugates (ADCs) of the present invention are those of formula VII below.

Where
m is a number from 0 to 2;
n is 0 or 1;
X represents —C (═O) —NH ₂ or —COOH;
La represents _a self-destructing linker;
L _c represents a linker,
A1 is _one of the amino acids Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His. A residue from one of the following;
A ₂ is an amino acid Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent A residue from one of the N-alkyl-amino acids, preferably one of the N-methyl-amino acids (if there is more than one P3, P3 can have different meanings);
D1 is KSP according to formula II or IIa (ie without linker L);
R represents Z ₁ — (C═O) _q —,
q is 0 or 1,
Z ₁ represents C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _6-10-. Aryl-, C _5-10 -heterocyclic alkyl-, heteroaryl-, heteroaryl-alkyl-, C _5-10 -heteroaryl-alkoxy-, C _{1-10 -alkoxy-} , C _{6-10 -aryloxy-} or _{C 6-10} - aralkoxy _{-, C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _- represents a heterocyclic alkoxy group, it is _{-, C 5-10} - NH _2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -S (=} O ) ₂ -NH ₂ , —S (═O) ₂ —N (alkyl) ₂ , —COOH, —C (═O) —NH ₂ , —C (═O) —N (alkyl) ₂ , —OH, — May be substituted one or more times by H or a group —O _x — (CH ₂ CH ₂ O) _y —R ¹ ,
x is 0 or 1,
v is a number from 1 to 20,
R ¹ is -H, - alkyl (preferably _{C 1-12} - _alkyl), - _CH 2 -COOH, represents _-CH 2 _-CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2,
AB represents an antibody,
s is a number from 1 to 20, preferably from 2 to 8, particularly preferably from 2 to 4, for example 4.

Preferred antibody prodrug conjugates (APDCs) of the present invention are those of formula IX below.

Where
m is 0, 1 or 2;
n is 0 or 1;
X represents —C (═O) —NH ₂ or —COOH;
La represents _a self-destructing linker;
L _b represents a linker;
A1 is _one of the amino acids Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His. A residue from one;
A ₂ is an amino acid Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent A residue from one of the N-alkyl-amino acids, preferably one of the N-methyl-amino acids (if there is more than one P3, P3 can have different meanings);
D ₁ is a KSP according to Formula II or IIa (ie, no linker L);
R represents Z ₁ — (C═O) _q —,
q is 0 or 1,
Z ₁ represents C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _6-10-. Aryl-, C _5-10 -heterocyclic alkyl-, heteroaryl-, heteroaryl-alkyl-, C _5-10 -heteroaryl-alkoxy-, C _{1-10 -alkoxy-} , C _{6-10 -aryloxy-} or _{C 6-10} - aralkoxy _{-, C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _- represents a heterocyclic alkoxy group, it is _{-, C 5-10} - NH _2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -S (=} O _{) 2 -NH 2, -S (=} O) 2 -N ( _{alkyl) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _{alkyl) 2} or -OH,, -H or a group _{-O x - (CH 2 CH 2} O) at y -R ¹ may optionally be substituted one or more times,
x is 0 or 1,
v is a number from 1 to 20,
R ¹ is -H, - alkyl (preferably _{C 1-12} - _alkyl), - _CH 2 -COOH, represents _-CH 2 _-CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2,
AB represents an antibody,
s is a number from 1 to 20, preferably from 2 to 8, particularly preferably from 2 to 4, for example 4.

Linkers The literature discloses a variety of options for covalently coupling organic molecules to sites, such as antibodies, covalently (eg, Sochaj et al., Biotechnology). Advanceds, see a recent paper (2015), Panowski et al., MAbs 6 , 34-45 (2014)). Preferred according to the present invention is the binding of the KSP inhibitor to the antibody via the acceptor glutamine residue of the antibody using transglutaminase. Such acceptor glutamines can be introduced by manipulation of the antibody by mutation or by formation of a deglycosylated antibody. The number of acceptor glutamine residues in the antibody is preferably 2 or 4. A linker is used for coupling. Linkers can be divided into groups of in vivo cleavable linkers and in vivo stable linkers (see L. Ducry and B. Stamp, Bioconjugate Chem. 21 , 5-13 (2010)). ). An in vivo cleavable linker has a group that is cleavable in vivo, where it is between a group that is chemically cleavable in vivo and a group that is enzymatically cleavable in vivo. It is possible to distinguish. “In vivo chemically cleavable” and “in vivo enzymatically cleavable” are those in which the linker or group is stable in the circulation, in the target cell or only in the target cell. Cleavage by chemically or enzymatically different environments (relatively low pH; high glutathione concentrations; presence of lysosomal enzymes such as legumain, cathepsin or plastin, or glyosidases such as β-glucuronidases) Means release of a low molecular weight KSP inhibitor or derivative thereof. In vivo chemically cleavable groups are in particular disulfides, hydrazones, acetals and aminals; enzymatic in vivo cleavable groups are in particular 2-8-oligopeptide groups, in particular Dipeptide groups or glycosides. Peptide cleavage sites are described in Bioconjugate Chem. 2002, 13, 855-869, and Bioorganic & Medicinal Chemistry Letters 8 (1998) 3341-3346, as well as Bioconjugate Chem. 1998, 9, 618-626. These include, for example, valine-alanine, valine-lysine, valine-citrulline, alanine-lysine and phenylalanine-lysine (where appropriate with further amide groups).

  In vivo stable linkers are distinguished by high stability (less than 5% metabolites after 24 hours in plasma) and have no in vivo chemically or enzymatically cleavable groups as described above.

The linker -L- is preferably
The following basic structures (i) to (iv):
(I)-(C = O) _m -SG1-L1-L2-
(Ii)-(C = O) _m -L1-SG-L1-L2-
(Iii)-(C = O) _m -L1-L2-
(Iv)-(C = O) _m -L1-SG-L2
One of
m is 0 or 1; SG is a group that is cleavable in vivo (chemically or enzymatically), in particular disulfides, hydrazones, acetals and aminals; or 2 to 8 oligopeptides cleavable by proteases SG1 is an oligopeptide group or preferably a dipeptide group, L1 represents an in vivo stable organic group, and L2 represents a coupling group for the binder.

Particularly preferred according to the invention is the basic linker structure (iii), especially when the binder is an antibody. Metabolism results in the administration of the conjugate according to the invention having the basic linker structure (iii) and the coupling of the linker to the glutamine residue of a binder protein or peptide using transglutaminase yields a glutamine derivative of the formula

  Where L1 is in each case a low molecular weight KSP inhibitor such as formula (I), (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId) , (IIIe) or (IV) to (IX).

According to the present invention, L1 is preferably represented by the following formula:

Where
R ¹⁰ represents H, —NH ₂ or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C— (═O) —NH— or

(G1 is —NH—C (═O) — or

R ¹⁰ is preferably not NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G3 represents an optionally substituted linear or branched hydrocarbon chain and / or a linear and / or branched and / or cyclic alkylene group having 1 to 100 carbon atoms from a bond or an arylene group. , It is a group —O—, —S—, —S (═O) —, —S (═O) ₂ —, —NR ^y —, —NR ^y —C (═O) —, —C (NH) NR ^{y -, - C (= O} ) -NR y -, - NR y NR y -, - S (= O) 2 -NR y NR y -, - C (= O) -NR y NR y - (R y Represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, which are —NH—C (═O) —NH ₂ N, —COOH, respectively. , —OH, —NH ₂ , NH—CNNH ₂ , sulfonamide, sulfone , Sulfoxide or sulfonic acid may be substituted.), —C (═O) —, —CR ^x ═N—O— (R ^x represents H, C ₁ -C ₃ -alkyl or phenyl. ) And / or 4 or less N, O and S, —S (═O) — or —S (═O) ₂ —, a 3 to 10 membered aromatic or non-heteroatom having a heteroatom selected from the group consisting of Aromatic heterocycle (preferably

1 or the may be interrupted one or more times of) the hydrocarbon containing side _{chain, -NH-C (= O)} -NH 2, -COOH, -OH, -NH 2, NH-CNNH ₂ , may be substituted by sulfonamide, sulfone, sulfoxide or sulfonic acid.

G3 represents an optionally substituted linear or branched hydrocarbon chain and / or a linear and / or branched and / or cyclic alkylene group having 1 to 100 carbon atoms from a bond or an arylene group; It is a group —O—, —S—, —S (═O) —, —S (═O) ₂ —, —NH—, —C (═O) —, —NH—C (═O) —, — C (═O) —NH—, —NMe—, —NHNH—, —S (═O) ₂ —NHNH—, —C (═O) —NHNH— and N, O and S, or —S (═O 5- to 10-membered aromatic or non-aromatic heterocycle having 4 or fewer heteroatoms selected from the group consisting of

) May be interrupted one or more times by one or more, and when the side chain is present, it is —NH—C (═O) —NH ₂ , —COOH, —OH, —NH ₂ , NH— CNNH _2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid.

G3 preferably represents an optionally substituted linear or branched hydrocarbon chain and / or linear and / or branched and / or cyclic alkylene group having 1 to 100 carbon atoms from a bond or an arylene group. Represented by the groups —O—, —S—, —S (═O) —, —S (═O) ₂ —, —NH—, —C (═O) —, —NH—C (═O) —. , —C (═O) —NH—, —NMe—, —NHNH—, —S (═O) ₂ —NHNH—, —C (═O) —NHNH—, —CR ^x ═N—O— (R ^x represents H, C1-C3-alkyl or phenyl.) and 4 or less selected from the group consisting of N, O and S, -S (= O)-or -S (= O) _2- 3 to 10-membered aromatic or non-aromatic heterocycles having heteroatoms, such as 5 to 10-members (preferably Clause

Or

)) May be interrupted one or more times by one or more, and when a hydrocarbon chain containing a side chain is present, it is —NH—C (═O) —NH ₂ , —COOH, —OH, —NH _{2, -NH-CN-NH 2} , sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid.

Further interrupting groups in G3 are preferably

And
R ^x represents H, C ₁ -C ₃ -alkyl or phenyl.

Here, # ¹ is a bond to a KSP inhibitor, and # ² is a bond to a coupling group to a binder (for example, L2).

The linear or branched hydrocarbon chain of an arylene group and / or a linear and / or branched and / or cyclic alkylene group usually comprises an α, ω-divalent alkyl group having the specified number of carbon atoms . Examples which may be mentioned as preferred are methylene, ethane-1,2-diyl (1,2-ethylene), propane-1,3-diyl (1,3-propylene), butane-1,4-diyl. (1,4-butylene), pentane-1,5-diyl (1,5-pentylene), hexane-1,6-diyl (1,6-hexylene), heptane-1,7-diyl (1,7- Hexylene), octane-1,8-diyl (1,8-octylene), nonane-1,9-diyl (1,9-nonylene), decane-1,10-diyl (1,10-decylene). However, the alkylene group in the hydrocarbon chain can also be branched, i.e. one or more hydrogen atoms are C _1-10 straight-chain alkylene group mentioned above _- that is substituted by an alkyl group, a side chain Can be formed. The hydrocarbon chain may further comprise a cyclic alkylene group (cycloalkanediyl), for example 1,4-cyclohexanediyl or 1,3-cyclopentanediyl. These cyclic groups may be unsaturated. In particular, an aromatic group (arylene group) such as phenylene may be present in the hydrocarbon group. In the cyclic alkylene group and the arylene group, one or more hydrogen atoms may be substituted with a C _1-10 -alkyl group. In this way, a hydrocarbon chain which may be branched is formed. This hydrocarbon chain has a total of 0 to 100 carbon atoms, preferably 1 to 50, particularly preferably 2 to 25 carbon atoms.

If the side chain _is present, it is substituted -NH-C (= O) -NH 2, -COOH, -OH, -NH 2, -NH-CN-NH 2, sulfonamide, sulfone, by sulfoxide or sulfonic acid May be.

The hydrocarbon chain is a group —O—, —S—, —S (═O) —, —S (═O) ₂ —, —NH—, —C (═O) —, —NH—C (= O)-, -C (= O) -NH-, -NMe-, -NHNH-, -S (O) _2- NHNH-, -C (= O) -NHNH- and N, O and S, -S 5- to 10-membered aromatic or non-aromatic heterocycle having 4 or fewer heteroatoms selected from the group consisting of (═O) — or —S (O) ₂ — (preferably

) May be interrupted one or more times in the same or different manner.

Further interrupting groups in G3 are preferably

It is.

According to the present invention, L2 is preferably of the formula:

Represented by
p is 0 or 1;
q is 0 or 1;
G4 represents an optionally substituted alkyl or heteroalkyl chain, and any carbon of the chain is alkoxy, hydroxyl, alkylcarbonyloxy, -S-alkyl, thiol, -C (= O) -S-alkyl. , -C (= O) -NH- alkyl, -NH-C (= O) - alkyl, amine, -C (= O) may be substituted by -NH _2,
# ¹ represents the point of attachment to the group ^{L 1,}
# ² represents the binding site to the glutamine residue of the binder.

Preferably L2 is one of the following groups:

And
R ^y is —H, —C (═O) —NH-alkyl, —NH—C (═O) -alkyl, —C (═O) —NH ₂ , —NH ₂ ;
# ¹ represents the point of attachment to the group ^{L 1,}
# ² represents the binding site to the glutamine residue of the binder.

Preferably R ^y is —H or —NH—C (═O) —Me.

Preferably, the linker corresponds to the following formula:

Where
m is 0 or 1;
§ represents binding to the active compound molecule,
§§ represents binding to a binder peptide or protein,
L1 and L2 have the meanings provided above.

The linker referred to above is a complex of formula (I) or (II) in which the linker is coupled by substitution of a hydrogen atom at R ¹ or R ³ or in combination with a cleavable linker SG1 at R4. particularly preferred, i.e. ^{R 1} represents-L-# 1, or ^{R 3} represents-L-# 1, or ^{R 4} represents -SG1-L- # 1, # 1 represents a bond to the binder .

Preferred groups L1 in the above formula §- (C = O) _m -L1-L2-§§ are as follows, and in each case r is independently from 0 to 20, preferably from 0 to 15: Particularly preferred are numbers from 1 to 20, particularly preferably from 2 to 10.

Examples of conjugates with corresponding linkers have the following structure, X1, X2, X3, Ry and L1 have the meanings provided above, AK is an antibody bound to a binder, preferably a glutamine side chain N is 2 to 10, preferably 2 to 4, and preferably 2 or 4. Particularly preferably, AK is a human, humanized or chimeric monoclonal antibody or antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or antigen-binding fragment thereof or an anti-EGFR antibody or antigen-binding fragment thereof. When the binder is an antibody, it preferentially contains acceptor glutamine in the constant region. Such acceptor glutamines may be mutated to glutamine in a suitable position (eg mutated N297Q, Kabat EU numbering) or by the formation of deglycosylated or deglycosylated antibodies (eg enzymatic deglycosylation with PNGase F), Or by mutation of N297X, Kabat EU numbering). In the case of the latter deglycosylated or deglycosylated antibody, glutamine Q295 (Kabat EU numbering) becomes the acceptor glutamine. Highly preferred are antibodies comprising the mutation N297A or N297Q (Kabat EU numbering). Particularly preferred is an anti-TWEAKR antibody that specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169), particularly anti-TWEAKR antibody TPP-2090, or anti-Her2 antibody. All of the described antibodies include deglycosylated variants of these antibodies caused by deglycosylation with PNGaseF or by mutation of either heavy chain N297 (Kabat numbering) to any amino acid.

  Also preferred according to the invention is the basic structure (i), (ii) or (iv), in which SG1 or SG represents a group cleavable by cathepsin and L1 and L2 have the meanings provided above Have. Particularly preferred are the following groups:

-NH-Val-Ala-CONH- (this cleaves the amide bond at the C-terminal amide of alanine)
-NH-Val-Lys-CONH- (cleavage of amide bond at C-terminal amide of lysine)
-NH-Val-Cit-CONH- (cleavage of amide bond at C-terminal amide of citrulline)
-NH-Phe-Lys-CONH (cleavage of amide bond at C-terminal amide of lysine)
-NH-Ala-Lys-CONH- (cleavage of amide bond at C-terminal amide of lysine)
-NH-Ala-Cit-CONH- (cleavage of the amide bond at the C-terminal amide of citrulline).

SG1 or SG is particularly preferably

And
X represents —H or a C _1-10 -alkyl group, which is substituted by —NH—C (═O) —NH ₂ , —COOH, —OH, NH ₂ , —NH—CNNH ₂ or sulfonic acid. May be.

The table below provides examples of linker moieties -SG1-L1- or -L1-SG-L1-, where SG1 and SG are groups that can be cleaved by cathepsin. The L1 group is highlighted with a square. However, these groups L1 can be replaced by one of the groups L1 provided for the above formula §- (C = O) _m -L1-L2-§§.

Examples of complexes having structure (i) have the following structure, X1, X2, X3, R1 and Ry have the meanings provided above, and AK bound to a binder, preferably a glutamine side chain Represents an antibody, n is 2 to 10, preferably 2 to 4, and preferably 2 or 4. Particularly preferred is an anti-TWEAKR antibody, particularly anti-TWEAKR antibody TPP-2090, which specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169). All of the described antibodies include deglycosylated variants of these antibodies caused by deglycosylation with PNGaseF or by mutation of either heavy chain N297 (Kabat numbering) to any amino acid.

Production of KSP Inhibitor-Linker-Intermediates and Complexes A complex according to the present invention is produced by first providing a linker to a low molecular weight KSP inhibitor. Next, the intermediate thus obtained is reacted with a binder (preferably an antibody) using transglutaminase. Preferably, for site-specific coupling to the glutamine side chain, one of the following compounds is reacted with an acceptor glutamine-containing binder such as an antibody using transglutaminase.

In the formula, X ₁ , X ₂ , X ₃ , SG, L1, R1 and Ry have the same meaning as described above. In the above formula, instead of the free amino group (—NH ₂ ), the group R ²¹ — (C═O) ( _0-1) — (P3) _(0-2) —P ₂ —NH—CH (CH _{2) -C (= O) -NH 2)} -C (= O) -NH- is may be present ^(R 21, P2 and P3, for example, the formula (IIa) has the same meaning as defined above for .)

  When the binder is an antibody, it preferentially contains acceptor glutamine in the constant region. Such acceptor glutamines may be mutated to glutamine in a suitable position (eg, mutant N297Q, Kabat EU numbering) or by the formation of deglycosylated or deglycosylated antibodies (eg, enzymatic deglycosylation with PNGase F), Or by mutation of N297X, Kabat EU numbering). In the case of the latter deglycosylated or deglycosylated antibody, glutamine Q295 (Kabat EU numbering) becomes the acceptor glutamine. Highly preferred are antibodies comprising the mutation N297A or N297Q (Kabat EU numbering).

  The compound can be used, for example, in the form of its trifluoroacetate salt. For example, for reaction with antibodies, the compound is used in a 2 to 100-fold molar excess, more preferably a 10 to 100-fold molar excess, and even more preferably a 50 to 100-fold molar excess with respect to the binder.

  For intermediate coupling to the glutamine side chain, the reaction can be shown as follows.

Other intermediates and other antibodies can be reacted accordingly.

According to the invention, this provides the following complex:

In the above formula, X ₁ , X ₂ , X ₃ and R ₁ have the same meaning as in formula (II), and SG, R ^y and L 1 have the same meaning as above. AK is an antibody coupled to the glutamine side chain. Particularly preferably, AK1 is an anti-TWEAKR antibody, in particular an antibody that specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169), in particular the anti-TWEAKR antibody TPP-2090. All of the described antibodies include deglycosylated variants of these antibodies caused by deglycosylation with PNGaseF or by mutation of either heavy chain N297 (Kabat numbering) to any amino acid.

Particularly preferred KSP-inhibitor-complexes Particularly preferred are the following conjugates, wherein AK3a, AK3b, AK3d, AK3e represents a binder, preferably an antibody, n is 2 to 10, preferably 2 to 4, again Preferably 2 or 4 is represented. Preferred antibodies are those described below in section “BINDERs”, in particular antibody TPP-2090-HC-N297A (especially as AK3a), antibody TPP-2090-HC-N297Q (especially as AK3b), trastuzumab ( TPP-7510) (equivalent to trastuzumab-HC-N297A) (especially as AK3d), and trastuzumab (TPP-7511) (equivalent to trastuzumab-HC-N297Q) (especially as AK3e).

Definition of substituents
Alkylalkyl is 1 to 10, preferably 1 to 6 (C ₁ -C ₆ -alkyl), more preferably 1 to 4 (C ₁ -C ₄ -alkyl), particularly preferably 1 to 3 (C ₁ -C). ₃ -alkyl) represents a straight-chain or branched saturated monovalent hydrocarbon group having carbon atoms.

  Examples which may be mentioned as preferred are methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, isopropyl-, isobutyl-, sec-butyl, tert-butyl-, isopentyl-, 2-methylbutyl. -, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1 -Methylpentyl-, 2-ethylbutyl-, 1-ethylbutyl-, 3,3-dimethylbutyl-, 2,2-dimethylbutyl-, 1,1-dimethylbutyl-, 2,3-dimethylbutyl-, 1,3 -Dimethylbutyl-, 1,2-dimethylbutyl-. Particularly preferred are methyl, ethyl, propyl, isopropyl or tert-butyl groups.

Heteroalkylheteroalkyl represents alkyl as defined under “Alkyl” above, —O—, —S—, —NH—, —S (═O) —, —S (═O) ₂ —, —S It is interrupted by (= O) _2- NH-.

Cycloalkylcycloalkyl is 3 to 10 (C ₃ -C ₁₀ -cycloalkyl), preferably 3 to 8 (C ₃ -C ₈ -cycloalkyl), particularly preferably 3 to 7 (C ₃ -C ₇ -cyclo). Alkyl) represents a monocyclic saturated monovalent hydrocarbon group having carbon atoms.

  Examples of monocyclic cycloalkyl groups that may be mentioned as preferred are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

  Particularly preferred are cyclopropyl, cyclopentyl or cyclohexyl groups.

Aralkylaryl- C ₁ -C ₆ -alkyl- consists of an optionally substituted aryl group and a C ₁ -C ₆ -alkyl group, and the rest of the molecule via the C ₁ -C ₆ -alkyl group It is understood to mean a group attached to. Here, the alkyl group has the meaning provided above under alkyl.

  Examples that may be mentioned include benzyl, phenylethyl, phenylpropyl, phenylpentyl and the like, with benzyl being preferred.

Alkoxyalkoxy is 1 to 6 (C ₁ -C ₆ -alkoxy), preferably 1 to 4 (C ₁ -C ₄ -alkoxy), particularly preferably 1 to 3 (C ₁ -C ₃ -alkoxy) carbons. Represents a straight-chain or branched saturated alkyl ether group of the formula —O-alkyl having an atom.

  Examples which may be mentioned as preferred are methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentyloxy and n-hexyloxy.

Aralkoxy aralkoxy, substituted aryl groups and C ₁ -C be the 6 _- means a group attached to the rest of through an alkoxy group molecules _- is composed of an alkoxy group, C 1 _-C 6 Understood. Here, the alkoxy group has the meaning as defined above.

  Examples that may be mentioned include benzyloxy, phenylethyloxy, phenylpropyloxy, phenylpentyloxy, with benzyloxy being preferred.

Alkoxyalkylalkoxyalkyl represents an alkyl group substituted by alkoxy, for example C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl- or C ₁ -C ₃ -alkoxy-C ₁ -C ₃ -alkyl- It is.

Here, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl- means that the alkoxyalkyl group is bonded to the rest of the molecule via an alkyl moiety.

Heteroatom A heteroatom is understood to mean an oxygen, nitrogen or sulfur atom.

Arylaryl represents a monovalent monocyclic or bicyclic aromatic ring system consisting of carbon atoms. Examples include naphthyl- and phenyl-, preferred are phenyl- or phenyl groups.

Heteroarylheteroaryl represents a monovalent monocyclic or bicyclic aromatic ring system having 1, 2, 3 or 4 heteroatoms which may be the same or different. The heteroatom can be a nitrogen atom, an oxygen atom or a sulfur atom. The valence can be an aromatic carbon atom or a nitrogen atom.

  Monocyclic heteroaryl groups according to the invention have 5 or 6 ring atoms.

  Preferred mono are heteroaryl groups having 1 or 2 heteroatoms. Here, particularly preferred are one or two nitrogen atoms.

Heteroaryl groups having 5 ring atoms include, for example:
There are thienyl, thiazolyl, furyl, pyrrolyl, oxazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl and thiadiazolyl.

Heteroaryl groups having 6 ring atoms include, for example:
Examples include pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl.

  Bicyclic heteroaryl groups according to the invention have 9 or 10 ring atoms.

Heteroaryl groups having 9 ring atoms include, for example:
There are phthalidyl, thiophthalidyl, indolyl, isoindolyl, indazolyl, benzothiazolyl, benzofuryl, benzothienyl, benzimidazolyl, benzoxazolyl, azosinyl, indolizinyl, purinyl, indolinyl and the like.

Heteroaryl groups having 10 ring atoms include, for example, the following rings:
There are isoquinolinyl, quinolinyl, quinolidinyl, quinazolinyl, quinoxalinyl, cinnolinyl, phthalazinyl, 1,7- and 1,8-naphthyridinyl, pteridinyl, chromanyl and the like.

  Heteroaryl further has the meaning of partially saturated bicyclic aryl and partially saturated bicyclic heteroaryl.

Partially saturated bicyclic aryl and partially saturated bicyclic heteroaryl Partially saturated bicyclic aryl or heteroaryl groups have 4 to 7 ring atoms in each case through two immediately adjacent ring atoms. Consisting of a phenyl group or a monocyclic 5- or 6-membered heteroaryl group fused to an aliphatic cyclic group which may contain the same or different heteroatoms and may contain 1 or 2 heteroatoms Represents a bicyclic group. The heteroatom can be a nitrogen atom, an oxygen atom or a sulfur atom.

Examples of the partially saturated bicyclic aryl group include the following groups:
Examples include tetrahydronaphthyl, 2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl and 1,3-benzodioxolyl.

Examples of the partially saturated bicyclic heteroaryl group include the following groups:
5,6,7,8-tetrahydroquinolinyl- and 5,6,7,8-tetrahydroisoquinolinyl- and the like.

Heterocyclic alkyl Heterocyclic alkyl means monocyclic heterocycle, heterospirocycloalkyl and bridged heterocycle alkyl.

Monocyclic heterocycle Monocyclic heterocycle means a non-aromatic monocyclic ring system having 1, 2 or 3 heteroatoms which may be the same or different. The heteroatom can be a nitrogen atom, an oxygen atom or a sulfur atom.

  The monocyclic heterocyclic ring according to the invention can have 3 to 8, preferably 4 to 7, particularly preferably 5 or 6 ring atoms.

  For example, preferably the following: aziridinyl- is mentioned for monocyclic heterocyclic groups having 3 ring atoms.

  For example, preferably the following: azetidinyl-, oxetanyl- are mentioned for monocyclic heterocyclic groups having 4 ring atoms.

  For example, preferably the following: pyrrolidinyl-, imidazolidinyl-, pyrazolidinyl-, pyrrolinyl-, dioxolanyl- and tetrahydrofuranyl- are mentioned for monocyclic heterocyclic groups having 5 ring atoms.

  For example, preferably the following: piperidinyl-, piperazinyl-, morpholinyl-, dioxanyl-, tetrahydropyranyl- and thiomorpholinyl- are mentioned for monocyclic heterocyclic groups having 6 ring atoms.

  For example, preferably the following: azepanyl-, oxepanyl-, 1,3-diazepanyl-, 1,4-diazepanyl- are mentioned for monocyclic heterocyclic groups having 7 ring atoms.

  For example, preferably the following: oxocanyl-, azocanyl- are mentioned for monocyclic heterocyclic groups having 8 ring atoms.

  Among monocyclic heterocyclic groups, 4 to 7-membered saturated heterocyclic groups having 2 or less heteroatoms from the group consisting of O, N and S are preferred.

  Particularly preferred are morpholinyl-, piperidinyl- and pyrrolidinyl-.

One spiro cycloalkyl and heterocycloalkyl spirocycloalkyl any combination, two, and three or four carbon atoms replaced by a heteroatom as defined above and C ₅ -C 12 _- spiro cycloalkyl or C ₅ -C 12 _- heterocycloalkyl spiro cycloalkyl is understood to mean a two fused saturated ring system which share one common atom. Examples include spiro [2.2] pentyl, spiro [2.3] hexyl, azaspiro [2.3] hexyl, spiro [3.3] heptyl, azaspiro [3.3] heptyl, oxaazaspiro [3.3]. Heptyl, thiaazaspiro [3.3] heptyl, oxaspiro [3.3] heptyl, oxazaspiro [3.5] nonyl, oxazaspiro [3.4] octyl, oxazaspiro [5.5] undecyl, diazaspiro [3.3] heptyl, Thiazaspiro [3.3] heptyl, thiazaspiro [3.4] octyl, azaspiro [5.5] decyl, and further homologous spiro [3.4], spiro [4] variants modified by heteroatoms as defined .4], Spiro [5.5], Spiro [6.6], Spiro [2.4], Spiro [2.5], Spiro [2 6], spiro [3.5], spiro [3.6], spiro [4.5], there is a spiro [4.6] and spiro [5.6] system. Preferred is C ₆ -C ₁₀ -heterospirocycloalkyl-, for example, particularly preferred are 2-azaspiro [3.3] heptyl-, 1-thia-6-azaspiro [3.3] heptyl-2, -Thia-6-azaspiro [3.3] heptyl-, 2-oxa-6-azaspiro [3.3] heptyl-, 2,6-diazaspiro [3.3] heptyl-, 2-oxa-6-azaspiro [ 3.4] Octyl-, 2-oxa-6-azaspiro [3.5] nonyl-, 2-oxa-7-azaspiro [3.5] nonyl-, 8-azaspiro [4.5] decyl-, 2, 8-Diazaspiro [4.5] decyl-, 3-oxa-1,8-diazaspiro [4.5] decyl-.

C ₆ -C ₁₂ -bicycloalkyl or C _6-in which 1, 2, 3 or 4 carbon atoms are replaced by a heteroatom as defined above in any combination of bicycloalkyl and heterobicycloalkyl C _{12 -} heterobicycloalkyl is understood as meaning the condensation product of two saturated ring system that share two directly adjacent atoms. Examples include bicyclo [2.2.0] hexyl-, bicyclo [3.3.0] octyl-, bicyclo [4.4.0] decyl-, bicyclo [5.4.0] undecyl-, bicyclo [ 3.2.0] heptyl-, bicyclo [4.2.0] octyl-, bicyclo [5.2.0] nonyl-, bicyclo [6.2.0] decyl-, bicyclo [4.3.0] Nonyl-, bicyclo [5.3.0] decyl-, bicyclo [6.3.0] undecyl- and bicyclo [5.4.0] undecyl-, modified with heteroatoms, such as azabicyclo [ 3.3.0] octyl-, azabicyclo [4.3.0] nonyl-, diazabicyclo [4.3.0] nonyl-, oxazabicyclo [4.3.0] nonyl-, thiazabicyclo [4.3. 0] nonyl- or aza Cyclo [4.4.0] decyl -, and a group derived from further possible combinations according to the definition. Preference is given to C ₆ -C ₁₀ -heterobicycloalkyl, for example, especially preferred are perhydrocyclopenta [c] pyrrolyl-, perhydrofuro [3,2-c] pyridinyl-, perhydropyrrolo [1,2 -A] pyrazinyl-, perhydropyrrolo [3,4-c] pyrrolyl-.

Preferred examples of C ₆ -C ₁₂ -bicycloalkyl- are perhydronaphthalenyl- (decalinyl-), perhydrobenzoannulenyl-, perhydroazulenyl-, perhydroindanyl-, perhydropentalenyl. -.

Bridged cycloalkyl and crosslinking heterocyclealkyl crosslinking _C 6 _{-C 12} - cycloalkyl - or crosslinking _C 6 _{-C 12} - heterocycloalkyl - crosslinked _C 6 _{-C 12} ring system, such as the two atoms which are not adjacent to each other directly Is understood to mean the condensation of at least two saturated rings sharing This results in a bridged carbocycle (bridged cycloalkyl-) or a bridged heterocycle (bridged heterocycle) in which any one, two, three or four carbon atoms are replaced by a heteroatom as defined above in any combination. Alkyl-) can occur. Examples include bicyclo [2.2.1] heptyl-, azabicyclo [2.2.1] heptyl-, oxazabicyclo [2.2.1] heptyl-, thiazabicyclo [2.2.1] heptyl-, Diazabicyclo [2.2.1] heptyl-, bicyclo [2.2.2] octyl-, azabicyclo [2.2.2] octyl-, diazabicyclo [2.2.2] octyl-, oxazabicyclo [2. 2.2] octyl-, thiazabicyclo [2.2.2] octyl-, bicyclo [3.2.1] octyl-, azabicyclo [3.2.1] octyl-, diazabicyclo [3.2.1] octyl- Oxazabicyclo [3.2.1] octyl-, thiazabicyclo [3.2.1] octyl-, bicyclo [3.3.1] nonyl-, azabicyclo [3.3.1] nonyl-, diazabi Chlo [3.3.1] nonyl-oxazabicyclo [3.3.1] nonyl-, thiazabicyclo [3.3.1] nonyl-, bicyclo [4.2.1] nonyl-, azabicyclo [4.2 .1] nonyl-, diazabicyclo [4.2.1] nonyl-, oxazabicyclo [4.2.1] nonyl-, thiazabicyclo [4.2.1] nonyl-, bicyclo [3.3.2] decyl -, Azabicyclo [3.3.2] decyl-, diazabicyclo [3.3.2] decyl-, oxazabicyclo [3.3.2] decyl-, thiazabicyclo [3.3.2] decyl- or azabicyclo [ 4.2.2] Decyl- and there are further possible combinations by definition. Preference is given to bridged C ₆ -C ₁₀ -heterocyclic alkyl-, for example, particularly preferably 2-azabicyclo [2.2.1] heptyl-, 2,5-diazabicyclo [2.2.1] heptyl-, 2-oxa-5-azabicyclo [2.2.1] heptyl-, 8-azabicyclo [3.2.1] octyl-, 8-oxa-3-azabicyclo [3.2.1] octyl-, 3,9 -Diazabicyclo [4.2.1] nonyl-.

Halogenated alkyl group A halogenated alkyl group is an alkyl group having at least one halogen substituent. A halo-C ₁ -C ₆ -alkyl group is an alkyl group having 1 to 6 carbon atoms and at least one halogen substituent. If a plurality of halogen substituents are present, these may be different from each other. Preferred are fluoro _-C 1 -C ₆ - alkyl, fluoro _-C 1 -C ₄ - alkyl and fluoro _-C 1 -C ₃ - alkyl group. Examples which may likewise be mentioned as preferred are trifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 4,4,5,5,5-pentafluoropentyl or 3,3,4, 4,5,5,5-heptafluoropentyl group.

  Preference is given to perfluoroalkyl groups such as trifluoromethyl or pentafluoroethyl.

Further Definitions The term “transglutaminase” used interchangeably with “TGase” or “TG” is a primary amine that is structurally related to the γ-carboxamide group of peptide-bound glutamine, such as an aminopentyl group, for example It refers to an enzyme capable of cross-linking a protein by an acyl rearrangement reaction with the ε-amino group of peptide-bound lysine to form an 8- (y-glutamyl) lysine isopeptide bond. TGases include in particular bacterial transglutaminase (BTG), such as the enzyme having the EC reference number EC 2.3.2.13 (protein-glutamine-y-glutamyltransferase).

  The term “acceptor glutamine” when referring to an amino acid residue of an antibody is recognized by TGase under suitable conditions and is a reaction between glutamine and a lysine or structurally related primary amine, such as an aminopentyl group. Means a glutamine residue that can be cross-linked by TGase. Optionally, the acceptor glutamine is surface exposed glutamine.

  As used herein, “amino acid modification” or “mutation” means an amino acid substitution, insertion and / or deletion in a polypeptide sequence. A preferred amino acid modification herein is a substitution. As used herein, “amino acid substitution” or “substitution” refers to the replacement of an amino acid with another amino acid at a given position in the protein sequence. For example, substitution Y50W refers to a variant of the parent polypeptide in which tyrosine at position 50 is replaced by tryptophan. A “variant” of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or “parent” polypeptide. Polypeptide variants can have one or more amino acid substitutions, deletions and / or insertions at certain positions within the native amino acid sequence.

  The term “site-specific complex” refers to a binder, preferably an antibody, and a complex of moieties, preferably a linker-drug-moiety, which binder is in one or more predetermined positions of the binder, preferably a glutamine residue. Functionalized with groups. Transglutaminases (TGase), such as bacterial transglutaminase (BTG) (EC 2.3.2.13), exhibit strict specificity in the recognition of glutamine protein substrates and catalyze “site-specific complexation” Can do.

  The term “homogeneous complex” or “homogeneous ADC” refers to the composition of a site-specific complex in which at least 60, 70, 80 or 90% of the binder has the same number of complexing moieties per binder. In the case of antibodies, the number of complexing moieties per antibody will be even, preferentially 2 or 4.

Binder In the broadest sense, the term “binder” is understood to mean a molecule that binds to a target molecule present in certain target cell populations that are treated by the binder / active compound complex. The term binder is to be understood in its broadest sense and includes, for example, lectins, proteins capable of binding to certain sugar chains, and phospholipid binding proteins. Such binders include, for example, high molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptides (eg, Keefe AD., Et al., Nat. Rev. Drug Discov. 2010; 9: 537-550 aptamers (US 5,270,163)), or vitamins) and all other cell-binding molecules or substances. Binding proteins are, for example, antibodies comprising deglycosylated variants, and antibody fragments or antibody mimetics such as Affibodies, Adnectins, Anticarins, DARPins, Avimails, Nanobodies (Gebauer M. et. al., Curr. Opinion in Chem. Biol.2009; 13: 245-255; Nutall SD et al., Curr. Opinion in Pharmacology 2008; 8: 608-617). The binding peptide may be, for example, a ligand / receptor pair ligand, such as a ligand / receptor pair VEGF / KDR VEGF, such as a ligand / receptor versus transferrin / transferrin receptor transferrin or a cytokine / cytokine receptor, such as a ligand / receptor. Body versus TNFα of the TNFα / TNFα receptor.

  A “binder” comprises an acceptor glutamine residue that can be functionalized by a transglutaminase (TGase), such as a bacterial transglutaminase (BTG) (EC 2.3.2.13). The acceptor glutamine is natural or generated with no change in binder. Acceptor glutamine is a glutamine residue inserted at a suitable position (eg, via fusion to a label containing the acceptor glutamine), a mutation to a glutamine residue at a suitable position, an amino acid residue mutation (The mutation has the effect that a natural glutamine residue that was not previously recognized by TGase becomes an acceptor glutamine.) Or a change in post-translational modification (eg glycosylation) It is considered that a natural glutamine residue that has not been recognized by TGase has the effect of becoming an acceptor glutamine. When the binder is an antibody, it preferentially contains acceptor glutamine in the constant region. Such acceptor glutamines may be mutated to glutamine in a suitable position (eg, mutant N297Q, Kabat EU numbering) or by the formation of deglycosylated or deglycosylated antibodies (eg, enzymatic deglycosylation with PNGase F), Or by mutation of N297X, Kabat EU numbering). In the case of the latter deglycosylated or deglycosylated antibody, glutamine Q295 (Kabat EU numbering) becomes the acceptor glutamine. Highly preferred are antibodies comprising the mutation N297A or N297Q (Kabat EU numbering).

  As used herein, the term “deglycosylated antibody” or “deglycosylated antibody” or “deglycosylated antibody” refers to an antibody or antibody comprising an Fc region that does not have a glycan attached to a conserved N-linked site in the CH2 domain of the Fc region. Used to define derivatives. Deglycosylated antibodies can be produced, for example, by mutation of the heavy chain glycosylation site of N297 (using Kabat EU numbering) or by expression of the antibody in an expression system without glycosylation. Methods for enzymatic deglycosylation of antibodies are known in the art (eg, Winkelhave & Nicolson (1976), J Biol Chem. 251 (4): 1074-80). Deglycosylated antibodies can be produced, for example, by enzymatic deglycosylation using PNGaseF. In one embodiment of the invention, deglycosylated antibodies can be produced by antibody expression in a prokaryotic host. Suitable prokaryotic hosts include E. coli, Bacillus subtilis, Salmonella typhimurium and various species of Pseudomonas, Streptomyces and Staphylococcus. It is not limited to. In another embodiment of the invention, deglycosylation antibodies can be achieved using a mammalian expression system in conjunction with the glycosylation inhibitor tunicamycin (Nose & Wigell (1983), Proc Natl Acad Sci USA, 80 (21 ): 6632-6). That is, the modification is prevention of glycosylation at a conserved N-linked site in the CH2 domain of the Fc portion of the antibody.

  The literature also discloses various options for homogeneous site-specific covalent coupling (complexation) of organic molecules to antibodies. Preferred according to the invention is the binding of a venom to the antibody via two or four acceptor glutamine residues of the antibody.

  “Target molecule” in the broadest sense means a molecule that is present in a target cell population and can be a protein (eg, a growth factor receptor) or a non-peptide molecule (eg, a sugar or phospholipid). It is understood. It is preferably a receptor or an antigen.

  The term “extracellular” target molecule describes a target molecule that is extracellular and bound to a cell, or a portion of a target molecule that is extracellular. That is, the binder can bind to its extracellular target molecule on intact cells. The extracellular target molecule can be immobilized on the cell membrane or can be a component of the cell membrane. Those skilled in the art know how to identify extracellular target molecules. In the case of proteins, it can be done by confirming the transmembrane domain and the orientation of the protein in the membrane. These data are usually deposited in a protein database (eg SwissProt).

  The term “cancer target molecule” describes a target molecule that is more abundant on one or more cancer cell types than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell types relative to non-cancer cells of the same tissue type, and “selectively” is compared to non-cancer cells of the same tissue type. To explain that it is at least twice as abundant on cancer cells ("selective cancer target molecule"). By using a cancer target molecule, selective therapy of cancer cells using the complex according to the present invention becomes possible.

  The binder can be bound to the linker via a bond. The binding of the binder can be via the carbonyl functionality of the glutamine side chain. Preferably, such glutamine residues are recognized as substrates by bacterial transglutaminase. The glutamine residues according to the invention can be present in the natural binder or can be introduced by methods of molecular biology, for example by deglycosylation of the antibody by PNGase F or introduction of mutations. According to the present invention, binding of the binder to the poison is only marginally effective on the binding activity of the binder with respect to the target molecule. In a preferred embodiment, the binding has no effect on the binding affinity and specificity of the binder with respect to the target molecule.

According to the present invention, the term “antibody” is to be understood in its broadest sense and is an immunoglobulin molecule, such as an intact or modified monoclonal antibody, a deglycosylated antibody, a polyclonal antibody or a multispecific antibody (eg Bispecific antibodies). Immunoglobulin molecules preferably comprise molecules having four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), typically linked by disulfide bridges. Each heavy chain comprises a heavy chain variable domain (abbreviated VH) and a heavy chain constant domain. The constant domain of the heavy chain, for example, may include three domains CH1, CH ₂ and CH3. Each light chain includes a variable domain (abbreviated VL) and a constant domain. The constant domain of the light chain includes a domain (abbreviated CL). The VH and VL domains can be further subdivided into regions with hypervariability [also referred to as complementarity determining regions (abbreviated CDR)] and regions with low sequence variability (framework region, abbreviated FR). Typically, each VH and VL region consists of three CDRs and no more than four FRs. For example, the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies can be obtained from any suitable species, such as rabbits, llamas, camels, mice or rats. In one embodiment, the antibody is of human or mouse origin. The antibody can be, for example, human, humanized or chimeric.

  The term “monoclonal” antibody refers to an antibody obtained from a group of substantially homogeneous antibodies, ie, the individual antibodies of that group are identical except for spontaneous mutations that may be small. Monoclonal antibodies recognize single antigenic binding sites with high specificity. The term monoclonal antibody does not refer to a specific manufacturing process.

  The term “intact” antibody refers to an antibody that contains both light and heavy chain antigen-binding and constant domains. The constant domain can be a natural domain with a number of modified amino acid positions or variants thereof and can be deglycosylated.

  The term “modified intact” antibody refers to an intact antibody fused via an amino terminus or carboxy terminus by covalent linkage (eg, peptide linkage) with an additional polypeptide or protein that is not of antibody origin. In addition, antibodies can be modified to introduce reactive cysteines at defined positions to facilitate coupling to venoms (Junutula et al. Nat Biotechnol. 2008, Aug.,). 26 (8): 925-32).

  The term “human” antibody refers to an antibody that can be obtained from a human or is a synthetic human antibody. A “synthetic” human antibody is an antibody that can be partially or fully obtained in silico from synthetic sequences based on analysis of human antibody sequences. Human antibodies can be encoded, for example, by nucleic acids isolated from a library of antibody sequences of human origin. Examples of such antibodies are described in Soderlind et al. , Nature Biotech. 2000, 18: 853-856. Such “human” and “synthetic” antibodies also include deglycosylated variants formed by deglycosylation with PNGaseF or by mutation of the heavy chain to either amino acid of N297 (Kabat numbering). It is.

  The term “humanized” or “chimeric” antibody describes an antibody consisting of the non-human and human portions of the sequence. In these antibodies, part of the human immunoglobulin (acceptor) sequence is replaced by a non-human immunoglobulin (donor) sequence part. In many cases, the donor is a mouse immunoglobulin. In the case of a humanized antibody, the acceptor CDR amino acids are replaced by donor amino acids. In some cases, the amino acids of the framework are also replaced by the corresponding amino acids of the donor. In some cases, humanized antibodies comprise amino acids that were introduced during antibody optimization and that are not present in either the acceptor or the donor. In the case of a chimeric antibody, the donor immunoglobulin variable domain is fused to the constant region of a human antibody. Such “humanized” and “chimeric” antibodies also include deglycosylated variants formed by deglycosylation by PNGaseF or by mutation of the heavy chain to either amino acid of N297 (Kabat numbering). It is.

  As used herein, the term complementarity determining region (CDR) refers to the amino acids of a variable antibody domain that are required for binding to an antigen. Typically, each variable region has three CDR regions called CDR1, CDR2 and CDR3. Each CDR region can include amino acids according to the Kabat definition and / or hypervariable loop amino acids defined according to Cotia. The Kabat definition is, for example, approximately amino acid positions 24 to 34 (CDR1), 50 to 56 (CDR2) and 89 to 97 (CDR3) of the variable light chain and 31 to 35 (CDR1), 50 to 65 of the variable heavy chain. (CDR2) and the region from 95 to 102 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Inter, 5th Ed. Public Health Services, National Institute of Health, 19th National Institutes.). Cotia defines, for example, approximately amino acid positions 26 to 32 (CDR1), 50 to 52 (CDR2) and 91 to 96 (CDR3) of the variable light chain and 26 to 32 (CDR1) of the variable heavy chain, 53 to 55 ( CDR2) and the region from 96 to 101 (CDR3) (Chothia and Less; J Mol Biol 196: 901-917 (1987)). Optionally, the CDR can include amino acids from the CDR regions defined according to Kabat and Cotia.

  Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies can be divided into different groups. There are five main groups of intact antibodies: IgA, IgD, IgE, IgG and IgM, some of which are divided into further subgroups (isotypes), eg IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Can do. The heavy chain constant domains that correspond to the different groups are referred to as [alpha / α], [delta / δ], [epsilon / ε], [gamma / γ] and [mu (my) / μ]. Both the three-dimensional structure and subunit structure of antibodies are known.

  The term “functional fragment” or “antigen-binding antibody fragment” of an antibody / immunoglobulin is defined as an antibody / immunoglobulin fragment (eg, an IgG variable domain) that also contains the antigen-binding domain of an antibody / immunoglobulin. Is done. An “antigen binding domain” of an antibody typically includes one or more hypervariable regions of the antibody, eg, CDR, CDR2 and / or CDR3 regions. However, the “framework” or “backbone” region of the antibody may be involved in the binding of the antibody to the antigen. The framework region forms the CDR skeleton. Preferably, the antigen binding domain is at least amino acids 4 to 103 of the variable light chain and amino acids 5 to 109 of the variable heavy chain, more preferably amino acids 3 to 107 of the variable light chain and 4 to 111 of the variable heavy chain, particularly preferred Contains the fully variable light and heavy chains, ie amino acids 1 to 109 of VL and 1 to 113 of VH (numbering according to WO 97/08320).

“Functional fragments” or “antigen-binding antibody fragments” of the present invention include Fab, Fab ′, F (ab ′) 2 and Fv fragments, bispecific antibodies, single domain antibodies (DAbs), linear antibodies, Including, but not limited to, individual chains of antibodies (single chain Fv, abbreviated scFv); and multispecific antibodies such as those formed from bispecific antibodies and trispecific antibodies such as antibody fragments. (C. A. K Borrebaeck, editor (1995) Antibody Engineering in Molecular Biology), Oxford University & S & B, E. ringer Laboratory Manual), Springer Verlag). Antibodies other than “multispecific” or “multifunctional” antibodies are those that have the same binding site. Multispecific antibodies can be specific for different epitopes of an antigen or can be specific for epitopes of multiple antigens (eg, WO93 / 17715; WO92 / 08802; WO91 / 00360; WO92 / Tutt, et al., 1991, J. Immunol.147: 60 69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 920; 5,601,819; or Kostelny et al., 1992, J. Immunol. 148: 15471553). F (ab ′) ₂ or Fab molecules can be constructed so that the effects of intermolecular disulfide interactions that occur between the Ch1 and CL domains can be reduced or completely prevented.

  “Epitope” refers to a protein determinant that has the ability to specifically bind to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains or combinations thereof and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

  A “functional fragment” or “antigen-binding antibody fragment” is fused through its amino terminus or carboxyl terminus to another polypeptide or protein that does not originate from an antibody, by covalent bonds (eg, peptide linkages). Can be. In addition, antibodies and antigen-binding fragments can be modified by introducing reactive cysteines at defined locations to facilitate coupling to venoms (Junutula et al. Nat Biotechnol. 2008 Aug; 26 (8): 925-32).

  Polyclonal antibodies can be made by methods known to those skilled in the art. Monoclonal antibodies can be made by methods known to those skilled in the art (Koehler and Milstein, Nature, 256, 495-497, 1975). Human and humanized monoclonal antibodies can be made by methods known to those skilled in the art (Olsson et al., Meth Enzymol. 92, 3-16 or Cabilly et al US 4,816,567 or Boss et al US 4,816). 397).

  Those skilled in the art can, for example, use transgenic mice (N Lonberg and D Huszar, Int Rev Immunol. 1995; 13 (1): 65-93) or phage display technology (Clackson et al., Nature. 1991 Aug 15; 352 (6336). ): 624-8) knows various ways to make human antibodies and fragments thereof. The antibody of the present invention can be obtained, for example, from a recombinant antibody library comprising amino acid sequences of a large number of antibodies collected from a very large number of healthy volunteers. Antibodies can also be made by known recombinant DNA techniques. Antibody nucleic acid sequences can be obtained by routine sequencing or can be obtained from publicly accessible databases.

  An “isolated” antibody or binder is one that has been purified to remove other components of the cell. Cell fouling components that can interfere with diagnostic or therapeutic use are, for example, enzymes, hormones, or other peptide or non-peptide components of the cell. Preferred antibodies or binders are those that have been purified to a range of just over 95% by weight with respect to the antibody or binder (eg, as measured by the Raleigh method, UV-Vis spectral analysis or SDS capillary gel electrophoresis). In addition, antibodies that have been purified to the extent that at least 15 amino acids of the amino terminus or internal amino acid sequence can be determined, or purified to homogeneity (homogeneity is SDS-under reduced or non-reduced conditions). Detected by PAGE (detection can be determined by Coomassie Blau staining or preferably silver staining). However, antibodies are usually obtained by one or more purification steps.

The term “specific binding” or “specifically binds” refers to an antibody or binder that binds to a given antigen / target molecule. Specific binding of an antibody or binder typically describes an antibody or binder having an affinity of at least 10 ⁻⁷ M (as Kd value; ie preferably having a Kd value less than 10 ⁻⁷ M) ), The antibody or binder is more sensitive to a given antigen / target molecule than a non-specific antigen / target molecule (eg bovine serum albumin or casein) that is neither a given antigen / target molecule nor a very closely related antigen / target molecule. Has at least a 2-fold higher affinity for the molecule. The antibody is preferably at least 10 ⁻⁷ M (as Kd value; ie, preferably having a Kd value less than 10 ⁻⁷ M), preferably at least 10 ⁻⁸ M, more preferably 10 ⁻⁹ M to 10 ^Has an affinity in the range of ^-11 M. The Kd value can be measured, for example, by surface plasmon resonance spectroscopy.

The antibody-drug conjugates of the invention also exhibit these ranges of affinity. Its affinity is preferably little affected by drug binding (in general, its affinity is reduced by less than an order of magnitude, ie, for example, at most 10 ⁻⁸ M to 10 ⁻⁷ M).

  The antibodies used according to the invention are also notable for high selectivity. High selectivity exists because the antibodies of the invention have an affinity for the target protein that is at least 2-fold, preferably 5-fold, or more preferably 10-fold better than other independent antigens, such as human serum albumin. (Affinity can be measured, for example, by surface plasmon resonance spectroscopy).

  Furthermore, the antibodies of the invention used are preferably cross-reactive. In order to facilitate and better interpret preclinical tests, eg toxicity tests or activity tests (eg tests in xenograft mice), the antibodies used according to the invention only bind to the human target protein. It is advantageous if it also binds to a biological target protein in the organism used for the test. In one embodiment, the antibodies used in accordance with the present invention are cross-reactive with the target protein of at least one other species in addition to the human target protein. For toxicity and activity tests, it is preferred to use rodent, canine and non-human primate organisms. Preferred rodent species are mice and rats. Preferred non-human primates are rhesus monkeys, chimpanzees and cynomolgus monkeys.

  In one embodiment, the antibody used according to the invention is in addition to a human target protein, to a target protein of at least one other species selected from the group of species consisting of mice, rats and cynomolgus monkeys (Macaca fascicularis). Cross-reactive for. Particularly preferred are antibodies used according to the invention that are cross-reactive with at least the mouse target protein in addition to the human target protein. Preference is given to cross-reactive antibodies whose affinity for the target protein of said other non-human species differs by up to 50 times, in particular up to 10 times, from the affinity for the human target protein.

The target molecule to which the antibody binder, eg antibody or antigen-binding fragment thereof, directed against the cancer target molecule is preferably a cancer target molecule. The term “cancer target molecule” describes a target molecule that is more abundant on one or more cancer cell types than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell types relative to non-cancer cells of the same tissue type, and “selectively” is compared to non-cancer cells of the same tissue type. To explain that it is at least twice as abundant on cancer cells ("selective cancer target molecule"). By using a cancer target molecule, selective therapy of cancer cells using the complex according to the present invention becomes possible.

  Antibodies that are specific for an antigen, eg, a cancer cell antigen, are prepared by one of skill in the art by methods familiar to those skilled in the art (eg, recombinant expression) or commercially available methods (eg, from Merck KGaA, Germany). can do. Examples of known commercially available antibodies in cancer therapy are Erbitux® (cetuximab, Merck KGaA), Avastin® (bevacizumab, Roche) and Herceptin® (trastuzumab, Genentech). Trastuzumab is an IgG1κ recombinant humanized monoclonal antibody that binds with high affinity to the extracellular domain of the human epidermal receptor in a cell-based assay (Kd = 5 nM). The antibody is produced recombinantly in CHO cells. Any of these antibodies can also be produced as a deglycosylated variant of these antibodies formed by deglycosylation with PNGaseF or by mutation of the heavy chain N297 (Kabat numbering) to any amino acid.

  In a preferred embodiment, the target molecule is a selective cancer target molecule.

  In a particularly preferred embodiment, the target molecule is a protein.

  In one embodiment, the target molecule is an extracellular target molecule. In a preferred embodiment, the extracellular target molecule is a protein.

  Cancer target molecules are known to those skilled in the art. Examples of these are listed below.

  Examples of cancer target molecules are:

(1) EGF receptor (NCBI reference sequence NP_005219.2), SEQ ID NO: 213 (1210 amino acids):
> Gi | 29725609 | ref | NP_005219.2 | EGFR receptor precursor [Homo sapiens]
MRPSGTAGAALLALLAALCPASRA LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPC RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS IATGMVGALLLLLVVALGIGL FMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYR LMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA
The extracellular domain is marked by an underline.

(2) Mesothelin (SwissProt reference number Q13421-3), SEQ ID NO: 214 (622 amino acids):
> Sp | Q13421-3 | MSLN_HUMAN Mesothelin OS = Homo sapiens GN = MSLN isoform 2
MALPTARPLLGSCCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS
LSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLARLSEPPEDLDALPL
DLLFLNPDAFSGPGPACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLSEA
DVRALGGLACLDPGRFVAESAEEVLPLPLVSCPGPLDQDQQEAARAALQGGGPPPYGPPSTW
SVSTMDALRGLLPVLGQPIIRSIPQGIVAAARQRQSRDPSWRQPERTIRPRFRREVEKT
ACPSGKKAREIDLESLIKYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKKLDELY
PQGYPESVIQHLGLFLKMSPEDIRKWNVTLSLETLKALLEVNKGHEMSPQVATIDRFVK
GRGQLDKDDTLDTLAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLTCCDPRRQLDVLYPKA
RLAFQNMNGSEYFVKIQSFLGGAPPEDLKALSQQNVSSMDLATFMKLRTDAVLPLTVAEVQ
KLLGPHVEGLKAEEERHRPVRDWILRQRQDDDLTLGLGLQGGIPNGYLVLDSMQEALSGT
PCLLGPGPPVLTVALLALLLASTLA
Mesothelin is encoded by amino acids 296 to 598. Amino acids 37-286 encode a megakaryocyte enhancement factor. Mesothelin is fixed to the cell membrane via a GPI anchor and is localized extracellularly.

(3) Carbonic anhydrase IX (SwissProt reference number Q16790), SEQ ID NO: 215 (459 amino acids):
> Sp | Q16790 | CAH9_HUMAN carbonic anhydrase 9OS = Homo sapiens GN = CA9PE = 1 SV = 2
MAPLCPSPWLPLLIPAPAPPGLTVQLLLLSLLLLVPVHP QRLPRMQEDSPLGGGGSSGEDDPL
GEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLKLPTVEAPG
DPQEPQNNAHRDKDGDDQSHWRYGGDPPPWPRVSPACAGRFQSPVDIRPQLAAFCPALRPL
ELLGFQLPPLPELLRLRNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHT
VEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEEIA
EEGSETQVPLGLDALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVMLSAKQLHTLS
DTLWGPGDSRLQLNFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCLAAGD ILALVF
GLLFAVTSVAFLVQMRRQHRRGTKGGVSYRPAEVAETGA
The extracellular domain is marked by an underline.

(4) C4.4a (NCBI reference sequence NP_055215.2; synonymous LYPD3), SEQ ID NO: 216 (346 amino acids):
> Gi | 93004088 | ref | NP_055215.2 | ly6 / PLAUR domain-containing protein 3-precursor [Homo sapiens]
MDPARKAGAQAMIWTAGWLLLLLLRGGAQA LECYSCVQKADDGCSPNKMKTVKCAPGVDVCTEAVGAVETIHGQFSLAVRGCGSGLPGKNDRGLDLHGLLAFIQLQQCAQDRCNAKLNLTSRALDPAGNESAYPPNGVECYSCVGLSREACQGTSPPVVSCYNASDHVYKGCFDGNVTLTAANVTVSLPVRGCVQDEFCTRDGVTGPGFTLSGSCCQGSRCNSDLRNKTYFSPRIPPLVRLPPPEPTTVASTTSVTTSTSAPVRPTSTTKPMPAPTSQTPRQGVEHEASRDEEPRLTGGAAGHQDRSNSGQYPAKGGPQQPHNKGC VAPTAG AALLLAVAAGVLL
The mature extracellular domain is marked by an underline.

(5) CD52 (NCBI reference sequence NP_001794.2), SEQ ID NO: 217
> Gi | 68334230 | ref | NP_001794.2 | CAMPATH-1 antigen-precursor [Homo sapiens]
MKRFLFLLLTISLMVMVQIQTGLSGQNDTSQTSSPSASSNSISGIFLFFFVANAIIHLFCFS
(6) Her2 (NCBI reference sequence NP — 004439.2), SEQ ID NO: 218
> Gi | 54792096 | ref | NP_004439.2 | receptor tyrosine-protein kinase erbB-2 isoform a [homo sapiens]
MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEK SKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVV GVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPL DSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV
(7) CD20 (NCBI reference sequence NP_068769.2), SEQ ID NO: 219
> Gi | 23110987 | ref | NP_068769.2 | B-lymphocyte antigen CD20 [Homo sapiens]
MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP
(8) Lymphocyte activation antigen CD30 (SwissProt ID P28908), SEQ ID NO: 220
> Gi | 68334811 | ref | NP_001234.2 | Tumor necrosis factor receptor superfamily member 8 isoform 1-precursor [Homo sapiens]
MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPMGLFPTQQCPQRPTDCRKQCEPDYYLDEADRCTACVTCSRDDLVEKTPCAWNSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMIVKFPGTAQKNTVCEPASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRLAQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYLDEAGRCTACVSCSRDDLVEKTPCAWNSSRTCECRPGMICATSATNSRARCVPYPICAAETVTKPQDMAEKDTTFEAPPLGTQP CNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK
(9) Lymphocyte adhesion molecule CD22 (SwissProt ID P20273), SEQ ID NO: 221
> Gi | 157168355 | ref | NP_001762.2 | B cell receptor CD22 isoform 1-precursor [Homo sapiens]
MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEP TVQILHSPAVEGSQVEFLCMSLANPLPTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACAACNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVKVQHSGAYWCQGTNSVG GRSPLSTLTVYYSPETIGRRVAVGLGSCLAILILAICGLKLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH
(10) Bone marrow cell surface antigen CD33 (SwissProt ID P20188), SEQ ID NO: 222
> Gi | 130979981 | ref | NP_001763.3 | bone marrow cell surface antigen CD33 isoform 1-precursor [homo sapiens]
MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVE DEELHYASLNFHGMNPSKDTSTEYSEVRTQ
(11) Transmembrane glycoprotein NMB (SwissProt ID Q14956), SEQ ID NO: 223
> Gi | 52669452 | ref | NP_001005340.1 | transmembrane glycoprotein NMB isoform a-precursor [Homo sapiens]
MECLYYFLGFLLLAARLPLDAAKRFHDVLGNERPSAYMREHNQLNGWSSDENDWNEKLYPVWKRGDMRWKNSWKGGRVQAVLTSDSPALVGSNITFAVNLIFPRCQKEDANGNIVYEKNCRNEAGLSADPYVYNWTAWSEDSDGENGTGQSHHNVFPDGKPFPHHPGWRRWNFIYVFHTLGQYFQKLGRCSVRVSVNTANVTLGPQLMEVTVYRRHGRAYVPIAQVKDVYVVTDQIPVFVTMFQKNDRNSSDETFLKDLPIMFDVLIHDPSHFLNYSTINYKWSFGDNTGLFVSTNHTVNHTYVLNGTFSLNLTVKAAAPGPCPPPPPPPRPS PTPSLATTLKSYDSNTPGPAGDNPLELSRIPDENCQINRYGHFQATITIVEGILEVNIIQMTDVLMPVPWPESSLIDFVVTCQGSIPTEVCTIISDPTCEITQNTVCSPVDVDEMCLLTVRRTFNGSGTYCVNLTLGDDTSLALTSTLISVPDRDPASPLRMANSALISVGCLAIFVTVISLLVYKKHKEYNPIENSPGNVVRSKGLSVFLNRAKAVFFPGNQEKDPLLKNQEFKGVS
(12) Adhesion molecule CD56 (SwissProt ID P13591), SEQ ID NO: 224
> Gi | 94420689 | ref | NP — 06606.3 | Neuron cell adhesion molecule 1 isoform 1 [Homo sapiens]
MLQTKDLIWTLFFLGTAVSLQVDIVPSQGEISVGESKFFLCQVAGDAKDKDISWFSPNGEKLTPNQQRISVVWNDDSSSTLTIYNANIDDAGIYKCVVTGEDGSESEATVNVKIFQKLMFKNAPTPQEFREGEDAVIVCDVVSSLPPTIIWKHKGRDVILKKDVRFIVLSNNYLQIRGIKKTDEGTYRCEGRILARGEINFKDIQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAEGFPEPTMSWTKDGEQIEQEEDDEKYIFSDDSSQLTIKKVDKNDEAEYICIAENKAGEQDATIHLKVFAKPKITYVENQTAMELEEQVTLTCEASG PIPSITWRTSTRNISSEEKTLDGHMVVRSHARVSSLTLKSIQYTDAGEYICTASNTIGQDSQSMYLEVQYAPKLQGPVAVYTWEGN
QVNITCEVFAYPSATISWFRDGQLLPSSNYSNIKIYNTPSASYLEVTPDSENDFGNYNCTAVNRIGQESLEFILVQADTPSSPSIDQVEPYSSTAQVQFDEPEATGGVPILKYKAEWRAVGEEVWHSKWYDAKEASMEGIVTIVGLKPETTYAVRLAALNGKGLGEISAASEFKTQPVQGEPSAPKLEGQMGEDGNSIKVNLIKQDDGGSPIRHYLVRYRALSSEWKPEIRLPSGSDHVMLKSLDWNAEYEVYVVAENQQGKSKAAHFVFRTSAQPTAIPANGSPTSGLSTGAIVGILIVIFVLLLVVVDITCYFLNKCGLFMCIAVNLCGKA PGAKGKDMEEGKAAFSKDESKEPIVEVRTEEERTPNHDGGKHTEPNETTPLTEPEKGPVEAKPECQETETKPAPAEVKTVPNDATQTKENESKA
(13) Surface molecule CD70 (SwissProt ID P32970), SEQ ID NO: 225
> Gi | 4507605 | ref | NP_001243.1 | CD70 antigen [homo sapiens]
MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP
(14) Surface molecule CD74 (SwissProt ID P04233), SEQ ID NO: 226
> Gi | 10835071 | ref | NP_004346.1 | HLA class II histocompatibility antigen gamma chain isoform b [homo sapiens]
MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM
(15) B-lymphocyte antigen CD19 (SwissProt ID P15391), SEQ ID NO: 227
> Gi | 296010921 | ref | NP_001171569.1 | B-lymphocyte antigen CD19 isoform 1-precursor [Homo sapiens]
MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRF KVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLAGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR
(16) Surface protein mucin-1 (SwissProt ID P15941), SEQ ID NO: 228
> Gi | 65301117 | ref | NP_002447.4 | mucin-1 isoform 1-precursor [homo sapiens]
MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNALSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAATSANL
(17) Surface protein CD138 (SwissProt ID P18827), SEQ ID NO: 229
> Gi | 29576886 | ref | NP_002988.3 | syndecan-1-precursor [homo sapiens]
MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQKPTKQEEFYA
(18) Integrin αV (Genbank accession number: NP — 000022.1.1), SEQ ID NO: 230
> Gi | 4504763 | ref | NP_002201.1 | integrin α-V isoform 1-precursor [homo sapiens]
MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFGFAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPIEFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDR SDGKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYPDLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDCGEDNVCKPKLEVSVDSDQKKIYIGDDNPLTLIVKAQNQGE AYEAELIVSIPLQADFIGVVRNNEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSSNLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHIFLPIPNWEHKENPETEEDVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPMNCTSDMEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIHTLGCGVAQCLKIVCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIEFPYKNLPIEDITNSTLVTTNVTWGIQPAPMPVPVWVIILA VLAGLLLLAVLVVFVMMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET
(19) Teratoma-derived growth factor 1 protein TDGF1 (Genbank accession number: NP_003203.1), SEQ ID NO: 231
> Gi | 4507425 | ref | NP_003203.1 | teratocarcinoma-derived growth factor 1 isoform 1-precursor [Homo sapiens]
MDCRKMARFSYSVIWIMAISKVFELGLVAGLGGHQEFARPSRGYLAFRDDSIWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCCLNGQTCMLGSFCCPPSFYGNRCGVCVCLPHDLCWLPK
(20) Prostate specific membrane antigen PSMA (Swiss Prot ID: Q04609), SEQ ID NO: 232
> Gi | 4758398 | ref | NP_004467.1 | glutamate carboxypeptidase 2 isoform 1 [homo sapiens]
MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGF GNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMND LMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA
(21) Tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317), SEQ ID NO: 233
> Gi | 32967311 | ref | NP_004422.2 | Ephrin type A receptor 2-precursor [Homo sapiens]
MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPH LTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGI GQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI
(22) Surface protein SLC44A4 (Genbank accession number: NP_001171515), SEQ ID NO: 234
> Gi | 295849282 | ref | NP_001171515.1 | choline transporter-like protein 4 isoform 2 [homo sapiens]
MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLVTFVLLLICIAYWAMT LYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKR K
(23) Surface protein BMPR1B (SwissProt: O00238)
(24) Transport protein SLC7A5 (SwissProt: Q01650)
(25) Epidermal prostate antigen STEAP1 (SwissProt: Q9UHE8)
(26) Ovarian cancer antigen MUC16 (SwissProt: Q8WXI7)
(27) Transport protein SLC34A2 (SwissProt: O95436)
(28) Surface protein SEMA5b (SwissProt: Q9P283)
(29) Surface protein LYPD1 (SwissProt: Q8N2G4)
(30) Endothelin receptor type B EDNRB (SwissProt: P24530)
(31) Ring finger protein RNF43 (SwissProt: Q68DV7)
(32) Prostate cancer-related protein STEAP2 (SwissProt: Q8NFT2)
(33) Cation channel TRPM4 (SwissProt: Q8TD43)
(34) Complement receptor CD21 (SwissProt: P20023)
(35) B cell antigen receptor complex-related protein CD79b (SwissProt: P40259)
(36) Cell adhesion antigen CEACAM6 (SwissProt: P40199)
(37) Dipeptidase DPEP1 (SwissProt: P16444)
(38) Interleukin receptor IL20Rα (SwissProt: Q9UHF4)
(39) Proteoglycan BCAN (SwissProt: Q96GW7)
(40) Ephrin receptor EPHB2 (SwissProt: P29323)
(41) Prostate stem cell-related protein PSCA (Genbank accession number: NP_005663.2)
(42) Surface protein LHFPL3 (SwissProt: Q86UP9)
(43) Receptor protein TNFRSF13C (SwissProt: Q96RJ3)
(44) B cell antigen receptor complex-related protein CD79a (SwissProt: P11912)
(45) Receptor protein CXCR5 (SwissProt: P32302)
(46) Ion channel P2X5 (SwissProt: Q93086)
(47) Lymphocyte antigen CD180 (SwissProt: Q99467)
(48) Receptor protein FCRL1 (SwissProt: Q96LA6)
(49) Receptor protein FCRL5 (SwissProt: Q96RD9)
(50) MHC class II molecule Ia antigen HLA-DOB (Genbank accession number: NP_002111.1)
(51) T cell protein VTCN1 (SwissProt: Q7Z7D3)
(52) TWEAKR (SEQ ID NO: 169 (protein); SEQ ID NO: 170 (DNA)
(53) Lymphocyte antigen CD37 (SwissProt: P11049)
(54) FGF receptor 2; FGFR2 (Gene ID: 2263; official symbol: FGFR2). The FGFR2 receptor occurs in various splice variants (α, β, IIIb, IIIc). All splice variants can serve as target molecules.

(55) Transmembrane glycoprotein B7H3 (CD276; Gene ID: 80381.
(56) B cell receptor BAFFR (CD268; Gene ID: 115650)
(57) Receptor protein ROR1 (Gene ID: 4919)
(58) Surface receptor IL3RA (CD123; Gene ID: 3561)
(59) CXC chemokine receptor CXCR5 (CD185; Gene ID 643)
(60) Receptor protein syncytin (Gene ID 30816).

  In a preferred subject of the invention, the cancer target molecule is selected from the group consisting of cancer target molecules (1) to (60), in particular (1), (6) and (52).

  In a further particularly preferred subject of the invention, the binder binds to an cancer target molecule (1) to (60), in particular an extracellular cancer target molecule selected from the group consisting of (1), (6) and (52). To do.

  In a further particularly preferred subject of the invention, the binder is specific for an extracellular cancer target molecule selected from the group consisting of cancer target molecules (1) to (60), in particular (1), (6) and (52). Join. In a preferred embodiment, the binder is internalized by the target cell as a result of its binding after its binding to its extracellular target molecule on the target cell. This causes the binder / active compound complex, which can be an immune complex or ADC, to be taken up by the target cell. The binder is then preferably treated intracellularly, preferably with lysosomes.

  In one embodiment, the binder is a binding protein. In preferred embodiments, the binder is an antibody, deglycosylated antibody, antigen-binding antibody fragment, multispecific antibody or antibody mimic.

  Preferred antibody mimetics are Affibodies, Adnectins, Anticalins, DARPins, Avimails, or Nanobodies. Preferred multispecific antibodies are bispecific and trispecific antibodies.

  In preferred embodiments, the binder is an antibody or antigen-binding antibody fragment, more preferably an isolated antibody or an isolated antigen-binding antibody fragment.

  Preferred antigen-binding antibody fragments are Fab, Fab ′, F (ab ′) 2 and Fv fragments, bispecific antibodies, DAbs, linear antibodies and scFv. Particularly preferred are Fabs, bispecific antibodies and scFv.

  In a particularly preferred embodiment, the binder is an antibody. Particularly preferred are monoclonal antibodies or antigen-binding antibody fragments thereof. Further particularly preferred are human, humanized or chimeric antibodies or antigen-binding antibody fragments thereof.

  Antibodies or antigen-binding antibody fragments that bind to a cancer target molecule can be made by those skilled in the art using known methods such as chemical synthesis or recombinant expression. Binders for cancer target molecules are commercially available or can be made by those skilled in the art using known methods such as chemical synthesis or recombinant expression. Further methods for producing antibodies or antigen-binding antibody fragments are described in WO2007 / 070538 (see “Antibodies” on page 22). One skilled in the art knows how to collect and use processes such as phage display libraries (eg Morphosys HuCAL Gold) to discover antibodies or antigen-binding antibody fragments (WO 2007/070538, pages 24 and onwards). (See AK Example 1 on page 70, AK Example 2 on page 72). A further method for obtaining antibodies using a DNA library from B cells is described, for example, on page 26 (WO 2007/070538). The process for humanized antibodies is described on pages 30-32 of WO2007070538 and is described by Queen, et al. , Pros. Natl. Acad. Sci. USA 86: 10029-10033, 1989 or WO 90/0786. In addition, methods for recombinant expression of proteins in general and in particular antibodies are known to those skilled in the art (see, eg, Berger and Kimrnel (Guide to Molecular Cloning Technologies, Methods in Enzymology, Vo. 152, Academic Inc., Academic Inc.). et al., (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N. Y .; ol) Vol.1-3, Vol.1-3, Vol.1-3. ogy, (F. M. Ausebel et al. [Eds.], Current Protocols, Green Publishing Associates, Inc./John Wiley & Sons, Inc.), Hallow et al. Harbor Laboratory Press (19881, Paul [Ed.]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al., (Using AntiLabs: US) (see manual, Cold Spring Harbor Laboratory Press (1998)), those skilled in the art know the corresponding vectors, promoters, and signal peptides required for protein / antibody expression.The normal process is 41-41 of WO2007 / 070538. It is also described in page 45. A method for obtaining an IgG1 antibody is described, for example, in Example 6 on page 74 of WO 2007/070538, allowing determination of internalization of the antibody after binding to an antigen. Processes are known to those skilled in the art and are described, for example, on page 80 of WO2007 / 070538. One skilled in the art can use the method described in WO2007 / 070538, which is used to make carbonic anhydrase IX (Mn) antibodies in a manner similar to the production of antibodies with different target molecule specificities.

Examples of antibodies that bind to the anti-EGFR antibody cancer target molecule EGFR are cetuximab (INN number 7906), panitumumab (INN number 8499) and nimotuzumab (INN number 8545). Cetuximab (Drug Bank accession number DB00002) is produced in SP2 / 0 mouse myeloma cells and is used in ImClone Systems Inc / Merck KgaA / Bristol-Myers Squibb Co. Chimeric anti-EGFR1 antibody sold by Cetuximab is indicated for the treatment of metastatic, EGFR-expressing colorectal cancer with the wild type K-Ras gene. It has an affinity of 10 ⁻¹⁰ M.

Array:
Cetuximab light chain (κ), SEQ ID NO: 235:
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Cetuximab heavy chain, SEQ ID NO: 236:
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Panitumumab (INN number 8499) (Drug Bank accession number DB01269) is a recombinant monoclonal human IgG2 antibody that specifically binds to human EGF receptor 1 sold by Abgenix / Amgen. Panitumumab originates from immunization of a transgenic mouse (XenoMouse). These mice can produce human immunoglobulins (light and heavy chains). A specific B cell clone producing an antibody against EGFR has been selected and this clone has been immortalized by CHO cells (Chinese hamster ovary cells). These cells are currently used to produce 100% human antibodies. Panitumumab is indicated for the treatment of EGFR-expressing metastatic colorectal cancer that is resistant to chemotherapy with fluoropyrimidine, oxaliplatin and irinotecan. It ^has an affinity of ^{10 -11} M.

Array:
Panitumumab light chain (κ), SEQ ID NO: 237:
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Panitumumab heavy chain, SEQ ID NO: 238:
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
Nimotuzumab (INN number 8545) (EP00586002, EP00712863) specifically binds to human EGF receptor 1 and is produced by YM BioScienecs Inc. Humanized monoclonal IgG1 antibody sold by (Mississauga Canada). It is produced in nonsecretory NSO cells (mammalian cell lines). Nimotuzumab is approved for the treatment of head and neck tumors, highly malignant astrocytomas and glioblastoma multiforme (unapproved in the EU and US) and pancreatic cancer (orphan drug, EMA). It has an affinity of ^{10 -8} M.

Nimotuzumab light chain, SEQ ID NO: 239:
DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Nimotuzumab heavy chain, SEQ ID NO: 240:
QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

  Further embodiments of EGFR antibodies are as follows.

Salzumumab / 2F8 / HuMax-EGFr, from Genmab A / S (WO02 / 100348, WO2004 / 056847, INN number 8605)
Nesitumumab / 11F8, ImClone / IMC-11F8, ImClone Systems Inc. From [Eli Lilly & Co] (WO2005 / 090407 (EP0173348-A1, US2007 / 0264253-A1, US7,598,350, WO2005 / 090407-A1), INN number 9083)
Matsuzumab / anti-EGFR MAb, Merck KGaA / anti-EGFR MAb, Takeda / EMD72000 / EMD-6200 / EMD-72000 and EMD-55900 / MAb425 / monoclonal antibody 425, from Merck KGaA / Takeda (WO92 / 15683, INN number 8103) ))
RG-7160 / GA-201 / GA201 / R-7160 / R7160 / RG7160 / RO-4858696 / RO-5083945 / RO4858696 / RO5083945, from Glycart Biotechnology AG (Roche Holding AG) (WO2010 / 112413-A11, WO2010 / 11 )
● GT-MAB5.2-GEX / CeteuEX, Glycotope GMBH (from WO2008 / 0286686-A2 (EP01900750-A1, EP01917666-A1, EP020833842-A2, US2010 / 0028947-A1))
● From ISU-101, Isu Abxis Inc (ISU Chemical Co Ltd) / Scancell (WO2008 / 004834-A1)
ABT-806 / mAb-806 / ch-806 / anti-EGFR monoclonal antibody 806, from Ludwig Institute for Cancer Research / Abbott / Life Science Pharmaceuticals (WO02 / 092771, WO2005WO02 / 0802)
SYM-004 (consisting of two chimeric IgG1 antibodies (992 and 1024)), Symphogen A / S (WO2010 / 022736-A2)
MR1-1 / MR1-1KDEL, IVAX Corp (Teva Pharmaceutical Industries Ltd) (Duke University), (Patent: WO2001 / 062931-A2)
Antibody against deletion mutant, from EGFRvIII, Amgen / Abgenix (WO2005 / 010151, US7,628,986)
● SC-100, from Scancell Ltd (WO01 / 088138-A1)
MDX-447 / EMD82633 / BAB-447 / H447 / MAb, EGFR, Medarex / Merck KgaA, Bristol-Myers Squibb (US) / Merck KGaA (DE) / Takeda (JP), (WO91 / 05871, WO92 / 15683) )
● Anti-EGFR-Mab, from Xencor (WO2005 / 056606)
DXL-1218 / anti-EGFR monoclonal antibody (cancer), Pharmaprojects PH048638 from InNexus, InNexus Biotechnology Inc.

  In a preferred embodiment, the anti-EGFR antibody is cetuximab, panitumumab, nimotuzumab, saltumumab, nesitumumab, matuzumab, RG-716, GT-MAB5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, Selected from the group consisting of SC-100, MDX-447 and DXL-1218.

  In a particularly preferred embodiment, the anti-EGFR antibody is selected from the group consisting of cetuximab, panitumumab, nimotuzumab, saltumumab, nesitumumab and matuzumab.

  One skilled in the art knows the methods that can be used to produce additional antibodies from the CDR regions of the above antibodies by sequence changes, and these additional antibodies have similar or better affinity for the target molecule. And / or have specificity. In addition, one of ordinary skill in the art knows methods that can be used to produce additional antibodies from the CDR regions of the antibodies by sequence changes, and these additional antibodies are deglycosylated and / or trans Modified to include one or more acceptor glutamine residues for glutaminase (TGase) catalysis.

  In a further embodiment, the anti-EGFR antibody or antigen-binding antibody fragment comprises the following antibodies: cetuximab, panitumumab, nimotuzumab, saltumumab, nesitumumab, matuzumab, RG-716, GT-MAB5.2-GEX, ISU-101, ABT-806. , SYM-004, MR1-1, SC-100, MDX-447 and DXL-1218 consisting of an antibody or antigen-binding antibody fragment comprising three CDR regions of the light chain and three CDR regions of the heavy chain Selected from the group.

  In a further embodiment, the anti-EGFR antibody or antigen-binding antibody fragment comprises three CDR regions of one light chain and three CDR regions of a heavy chain of the following antibodies: cetuximab, panitumumab, nimotuzumab, saltumumab, nesitumumab, matuzumab Selected from the group consisting of antibodies or antigen-binding antibody fragments. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Anti-carbonic anhydrase IX antibody Examples of antibodies that bind to the cancer target molecule carbonic anhydrase IX are described in WO2007 / 070538-A2 (eg claims 1 to 16).

  In a preferred embodiment, the anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment is an anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment 3ee9 (claim 4 (a) of WO2007 / 070538-A2), 3ef2 (WO2007 / 070538). -A2 claim 4 (b)), 1e4 (WO2007 / 070538-A2 claim 4 (c)), 3a4 (WO2007 / 070538-A2 claim 4 (d)), 3ab4 (WO2007 / 070538-A2) Claim 4 (e)), 3ah10 (WO2007 / 070538-A2 claim 4 (f)), 3bb2 (WO2007 / 070538-A2 claim 4 (g)), 1aa1 (WO2007 / 070538-A2) Item 4 (h)), 5a6 (WO2007 / 070538-A2) ) Is selected from the group consisting of 5Aa3 (claim 4 of WO2007 / 070538-A2 (j)).

Anti-C4.4a antibody:
According to the present invention, a C4.4a antibody can be used.

  Examples of C4.4a antibodies and antigen-binding fragments are described in WO2012 / 143499A2. By reference, all antibodies of WO2012 / 143499A2 are incorporated herein and can be used in the present invention. The sequence of the antibody is in Table 1 of WO2012 / 143499A2, in which each column indicates the individual CDR amino acid sequence of the variable light or variable heavy chain of the antibody listed in the first column.

  In one embodiment, an anti-C4.4a antibody or antigen-binding antibody fragment thereof is internalized by a cell after binding to a cell that expresses C4.4a.

  In a further embodiment, the anti-C4.4a antibody or antigen-binding antibody fragment comprises at least one, two or three CDR amino acid sequences of the antibodies listed in Table 1 of WO2012 / 143499A2 or Table 2 of WO2012 / 143499A2. Preferred embodiments of such antibodies are also listed in WO2012 / 143499A2 and are incorporated herein by reference.

Anti-HER2 antibody:
An example of an antibody that binds to the cancer target molecule Her2 is Trastuzumab (Genentech). Trastuzumab is a humanized antibody used in particular for the treatment of breast cancer.

  Further examples of antibodies that bind to HER2 include WO2009 / 123894-A2, WO200 / 8140603, in addition to trastuzumab (INN7637, CAS number: RN: 180288-69-1) and pertuzumab (CAS number 380610-27-5). An antibody disclosed in A2 or WO2011 / 044368-A2. An example of an anti-HER2 complex is trastuzumab-emtansine (INN number 9295). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Anti-CD20 antibody:
An example of an antibody that binds to the cancer target molecule CD20 is rituximab (Genentech). Rituximab (CAS number: 174722-31-7) is a chimeric antibody used for the treatment of non-Hodgkin lymphoma. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Anti-CD52 antibody:
An example of an antibody that binds to the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS number: 216503-57-0) is a humanized antibody used for the treatment of chronic lymphocytic leukemia. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Anti-mesothelin antibody:
Examples of anti-mesothelin antibodies are described, for example, in WO2009 / 068204. By reference, all antibodies described in WO2009 / 068204 are incorporated into the present description so that they can be used in the context of the invention disclosed herein.

  The anti-mesothelin antibodies used according to the invention are also noteworthy in invariants that preferably bind to mesothelin. Invariant binding is characterized, for example, by the fact that the antibody used according to the invention binds to an epitope of mesothelin that cannot be masked by further extracellular proteins. Such additional extracellular protein is, for example, the protein ovarian cancer antigen 125 (CA125). Preferably used antibodies are characterized in that their binding to mesothelin is not blocked by CA125.

Examples of antibodies that bind to the anti-CD30 antibody cancer target molecule CD30 and can be used to treat cancer, eg, Hodgkin lymphoma, are disclosed in brentuximab, iratumumab and WO2008 / 092117, WO2008 / 036688 or WO2006 / 089232. Antibody. An example of an anti-CD30 complex is brentuximab vedotin (INN number 9144). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to the anti-CD22 antibody cancer target molecule CD22 and can be used to treat cancer, eg, lymphoma, are inotuzumab and epratuzumab. Examples of anti-CD22 complexes are inotuzumab ozagamycin (INN number 8574) or anti-CD22-MMAE and anti-CD22-MC-MMAE (CASRN: 139504-50-0 and 474645-27-7, respectively) It is. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to the anti-CD33 antibody cancer target molecule CD33 and can be used to treat cancer, eg, leukemia, are gemtuzumab and lintuzumab (INN 7580). An example of an anti-CD33 complex is gemtuzumab-ozagamycin. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

An example of an antibody that binds to the anti-NMB antibody cancer target molecule NMB and can be used to treat cancer, such as melanoma or breast cancer, is Glenbatumumab (INN9199). An example of an anti-NMB complex is Glenbatumumab vedotin (CASRN: 474645-27-7). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

An example of an antibody that binds to the anti-CD56 antibody cancer target molecule CD56 and can be used to treat cancer, such as multiple myeloma, small cell lung cancer, MCC or ovarian cancer, is lorbotuzumab. An example of an anti-CD56 complex is lorbotuzumab mertansine (CASRN: 139504-50-0). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to the anti-CD70 antibody cancer target molecule CD70 and can be used to treat cancers such as non-Hodgkin's lymphoma or renal cell carcinoma are disclosed in WO2007 / 038637-A2 and WO2008 / 070593-A2. An example of an anti-CD70 complex is SGN-75 (CD70MMAF). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

An example of an antibody that binds to the anti-CD74 antibody cancer target molecule CD74 and can be used to treat cancer, eg, multiple myeloma, is miratuzumab. An example of an anti-CD74 complex is miratuzumab-doxorubicin (CASRN: 23214-92-8). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

An example of an antibody that binds to the anti-CD19 antibody cancer target molecule CD19 and can be used to treat cancer, eg, non-Hodgkin's lymphoma, is disclosed in WO 2008 / 031056-A2. Examples of further antibodies and anti-CD19 conjugates (SAR3419) are disclosed in WO2008 / 047242-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to the anti-mucin antibody cancer target molecule mucin-1 and can be used to treat cancer, eg, non-Hodgkin's lymphoma, are the antibodies disclosed in cribatuzumab and WO2003 / 106495-A2, WO2008 / 0286686-A2 . Examples of anti-mucin conjugates are disclosed in WO2005 / 009369-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to the anti-CD138 antibody cancer target molecule CD138 and complexes thereof and can be used to treat cancer, eg, multiple myeloma, are disclosed in WO2009 / 080829-A1, WO2009 / 080830-A1. Yes. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Anti-integrin-αV antibodies Examples of antibodies that bind to the cancer target molecule integrin αV and can be used to treat cancers such as melanoma, sarcoma or carcinoma are intatumumab (CASRN: 72535-28-4), abciximab (CASRN: 143653) -53-6), etaracizumab (CASRN: 892553-42-3) and US 7,465,449, EP719859-A1, WO2002 / 012501-A1 and WO2006 / 062779-A2. Examples of anti-integrin αV complexes are intetumumab-DM4 and other ADCs disclosed in WO2007 / 024536-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to anti-TDGF1 antibody cancer target molecule TDGF1 and can be used to treat cancer are disclosed in WO02 / 077033-A1, US7,318,924, WO2003 / 083041-A2 and WO2002 / 088170-A2. It is an antibody. An example of an anti-TDGF1 complex is disclosed in WO2002 / 088170-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to the anti-PSMA antibody cancer target molecule PSMA and can be used to treat cancer, eg, prostate cancer, are described in WO97 / 35616-A1, WO99 / 47554-A1, WO01 / 009192-A1 and WO2003 / 034903. The disclosed antibodies. Examples of anti-PSMA conjugates are disclosed in WO2009 / 026274-A1 and WO2007 / 002222. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to anti-EPHA2 antibody cancer target molecule EPHA2 and can be used in the production of conjugates and in the treatment of cancer are disclosed in WO 2004 / 091375-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to the anti-SLC44A4 antibody cancer target molecule SLC44A4 and can be used in the manufacture of conjugates and in the treatment of cancers such as pancreatic cancer or prostate cancer are disclosed in WO2009 / 033094-A2 and US2009 / 0175796-A1. ing. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

An example of an antibody that binds to an anti-HLA-DOB antibody cancer target molecule HLA-DOB is the antibody Lym-1 (CASRN: 301344-99-0) that can be used to treat cancer, eg, non-Hodgkin's lymphoma. An example of an anti-HLA-DOB complex is disclosed, for example, in WO2005 / 081711-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Examples of antibodies that bind to the anti-VTCN1 antibody cancer target molecule VTCN1 and can be used in the manufacture of conjugates and in the treatment of cancers such as ovarian cancer, pancreatic cancer, lung cancer or breast cancer are disclosed in WO2006 / 074418-A2. . By reference, these antibodies and antigen-binding fragments thereof are incorporated herein and can be used in the context of the present invention.

Anti-FGFR2 Antibody According to the present invention, an anti-FGFR2 antibody can be used.

  Examples of anti-FGFR2 antibodies and antigen binding fragments are described in WO20130186186. By reference, all the antibodies of WO201303186 are incorporated into the description of the invention and can be used in the invention. The sequences of the antibodies are shown in Tables 9 and 10 of WO20130186186. Preferred are antibodies referred to as M048-D01 and M047-D08, antigen-binding fragments and antibody variants derived therefrom. A preferred anti-FGFR2 binds to various splice variants termed FGFR2.

  In one embodiment, the anti-FGFR2 antibody or antigen-binding antibody fragment thereof is internalized by the cell after binding to the cell expressing FGFR2.

  In a further embodiment, the anti-FGFR2 antibody or antigen-binding antibody fragment comprises at least one, two or three CDR amino acid sequences of the antibodies listed in Table 9 or Table 10 of WO201303186. Preferred embodiments of such antibodies are likewise listed in WO201303186 and are hereby incorporated by reference.

Anti-TWEAKR Antibody In a preferred embodiment, when an anti-TWEAKR antibody or antigen-binding fragment thereof is used in the method according to the invention, the antibody or fragment is selected from: Further, antibodies that bind to TWEAKR are known to those skilled in the art, and refer to, for example, WO2015 / 189143 (A1), WO2014 / 198817 (A1), WO2009 / 020933 (A2) or WO2009140177 (A2). In addition, deglycosylated variants of the described anti-TWEAKR antibodies formed by deglycosylation with PNGaseF or by mutation of the heavy chain to either amino acid of N297 (Kabat numbering) are used in the method according to the invention. In addition, these antibody variants were modified to include one or more acceptor glutamine residues for transglutaminase (TGase) catalysis.

  The present invention particularly relates to an antibody that strongly induces apoptosis in various cancer cells overexpressing TWEAKR by causing strong activation of TWEAKR (SEQ ID NO: 169 (protein); SEQ ID NO: 170 (DNA)) or antigen-binding property thereof It relates to a complex with an antibody fragment or a variant thereof.

  The agonist activity of TWEAKR related to the induction of apoptosis and the inhibition of the growth of the aforementioned anti-TWEAKR antibodies (eg PDL-192) is limited and the efficacy of the endogenous ligand TWEAK is not reached. These antibodies bind to TWEAKR with a similar range of affinity compared to the endogenous ligand TWEAK (Michaelson JS et al., MAbs. 2011 Jul-Aug; 3 (4): 362-75; Cull PA et al., Clin Cancer Res. 2010 Jan 15; 16 (2): 497-508), and even antibodies with higher binding affinity do not necessarily show more effective signaling activity (Culp PA, et al., Clin Cancer Res. 2010 Jan 15; 16 (2): 497-508), this lack of agonist activity is not based on reduced affinity. Furthermore, it has been shown that the anti-tumor activity of the described antibodies is determined by Fc effector function, and it has been shown that ADCC plays an important role in in vivo efficacy in mouse models.

Formation of anti-TWEAKR antibodies Dimeric Fc fusion of human and mouse-TWEAKR as immobilization targets using a fully human antibody phage library (Hoet RM et al., Nat Biotechnol 2005; 23 (3): 344-8) The TWEAKR-specific human monoclonal antibody of the present invention was isolated by protein panning using the extracellular domain (Hoogenboom HR, Nat Biotechnol 2005; 23 (3): 1105-16). Eleven different Fab phages were identified and the corresponding antibody was cloned in a mammalian EgG expression vector providing a CH2-CH3 domain without soluble FAb. After identifying preferred antibodies, they were expressed as full-length IgG. Deglycosylated variants of the described antibodies were formed by introducing mutations N297A or N297Q in the heavy chains of individual antibodies. The construct is described, for example, in Tom et al. , Chapter 12, Methods Express: Expression Systems, editor Michael R. It was transiently expressed in mammalian cells according to the method described by Dyson and Yves Durocher, Scion Publishing Ltd, 2007 (see AK Example 1). The antibodies were purified by protein A chromatography and further characterized by binding affinity to soluble monomer TWEAKR using ELISA and BIAcore analysis according to the method described in AK Example 2. To confirm the cell binding properties of anti-TWEAKR antibodies, binding was examined by flow cytometry for many cell lines (HT29, HS68, HS578). An NFκB reporter gene assay was performed to examine the agonist activity of all 11 identified antibodies (human IgG1). The antibody with the highest in vitro activity (TPP-883) was selected for further activity and affinity maturation (see AK Example 1 for details). A single substitutional variant with improved agonist activity: CDR-H3 G102T was detected. Eventually, seven variants were selected based on their high affinity compared to the best single substitution variant G102T. Its corresponding DNA was cloned into a mammalian IgG expression vector and examined for functional activity in the NF-κB reporter gene assay described above. Eventually, the resulting sequences were compared to human germline sequences and adjusted for deviations that had little effect on affinity and potency. By antibody library screening and by affinity and / or activity maturation, the following antibodies: “TPP-2090”, “TPP-2149”, “TPP-2093”, “TPP-2148”, “TPP-2084”, “ “TPP-2077”, “TPP-1538”, “TPP-883”, “TPP-1854”, “TPP-1853”, “TPP-1857”, and “TPP-1858” were obtained.

  The antibodies of the present invention can be produced by methods known in the art such as antibody phage display screening (see, eg, Hoet RM et al., Nat Biotechnol 2005; 23 (3): 344-8), established hybridoma technology (eg, Koehler and Milstein Nature. 1975 Aug 7; 256 (5517): 495-7), or immunization of mice, especially immunization of hMAb mice (eg, Veloc immunized mice®).

Specific Embodiments of Anti-TWEAKR Antibodies One embodiment of the invention provides an antibody or antigen-binding antibody fragment thereof or variant thereof that exhibits strong induction of caspase 3/7 in one or more TWEAKR expressing cell lines (See also WO2015 / 189143A1 and WO2014 / 198817A1). In a preferred embodiment, one or more TWEAKR expressing cell lines are present in the group consisting of WiDr, A253, NCI-H322, HT29 and 786-O. “Induction of caspase 3/7” can be measured by common methods known in the art, such as those described herein. In one embodiment, “Induction of caspase 3/7” is used in accordance with the present invention using activity measurements with caspase 3/7 solution (Promega, # G8093) and luminescence readings with Victor V (Perkin Elmer). Ask. After the incubation period, caspase 3/7 activity was determined and the induction factor for caspase 3/7 was determined by comparison with untreated cells. An antibody has an induction factor greater than 1.2, preferably greater than 1.5, even more preferably greater than 1.8, even more preferably greater than 2.1, even more preferably greater than 2.5 It is said that it shows a “strong induction” of caspase 3/7. Compared to the previously described agonist antibodies [eg PDL-192 (TPP-1104), P4A8 (TPP-1324), 136.1 (TPP-2194)], and further compared to 300 ng / mL recombinant human TWEAK Thus, an anti-TWEAKR antibody is provided that has a stronger induction of caspase 3/7 in HT29 cells. This strong activity in caspase 3/7 induction in cancer cells was also observed in WiDr, A253, NIC-H322 and 786-O cells, in which case the antibodies of the present invention examined in most experiments were the reference antibodies A high coefficient of variation was induced compared to [PDL-192 (TPP-1104), P4A8 (TPP-1324)] and 300 ng / mL TWEAK. Some antibodies of the present invention bound to TWEAKR only with moderate affinity (> 10 nM), which was clearly less than the affinity of the endogenous ligand TWEAK and less than that of other known agonist antibodies. This property provides further possible advantages, for example the possibility of deeper penetration into the tumor.

  In this regard, one embodiment of the present invention is a novel epitope characterized in that it selectively binds to aspartic acid (D) at position 47 (D47) of TWEAKR (SEQ ID NO: 169; see also FIG. 1). It is to provide an antibody or an antigen-binding antibody fragment thereof that specifically binds to TWEAKR. The observed dependence for certain TWEAKR amino acids on antibody interactions correlates with the agonist activity measured in these antibodies. The natural ligand TWEAK shows an effective activation of TWEAKR and binds in response to leucine 46 in the cysteine-rich domain of TWEAKR (Pellegrini et al., FEBS 280: 1818-1829). P4A8 exhibits very low agonist activity and interacts at least partially with domains outside the cysteine-rich domain of TWEAKR. PLD-192 exhibits moderate agonist activity and binds to the cysteine-rich domain in response to R56, but is opposite to the TWEAK ligand site. The antibody of the present invention (for example, TPP-2090) binds according to D47, and TWEAK binds according to L46. Thus, TWEAK binds to similar but different binding sites (FIG. 7). Thus, antibodies of the invention that exhibit strong agonist activity bind to a novel epitope for antibodies that are associated with very high agonist activity (D47 dependent).

  The amino acid at position 47 (D47) of TWEAKR (SEQ ID NO: 169) is thought to be essential for binding of the antibody according to the invention, and this residue is converted to alanine as described in AK Example 2 and FIG. By changing, the antibody is more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 80%, more than 90% of its ELISA signal, Alternatively, losing 100% means that the antibody specifically binds to D at position 47 (D47) of TWEAKR (SEQ ID NO: 169). Alternatively, the antibody is more than 20% of its ELISA signal to TPP-2614 compared to TPP-2203, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, more than 70%, Alternatively, if more than 80%, or more than 90%, or 100% is lost, the antibody specifically binds to D at position 47 (D47) of TWEAKR (SEQ ID NO: 169). Preferably, when an antibody loses more than 80% of its ELISA signal to TPP-2614 compared to TPP-2203, the antibody specifically binds D at position 47 (D47) of TWEAKR (SEQ ID NO: 169). To do.

  In the present application, as shown in the following table, the following preferred antibodies of the present invention: “TPP-2090”, “TPP-2149”, “TPP-2093”, “TPP-2148”, “TPP-2084”, “TPP-2077”, “TPP-1538”, “TPP-883”, “TPP-1854”, “TPP-1853”, “TPP-1857”, “TPP-1858” are mentioned.

Table: Antibody protein sequences

  TPP-2090 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 2 and a light chain region corresponding to SEQ ID NO: 1.

  TPP-2658 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 241 and a light chain region corresponding to SEQ ID NO: 1.

  TPP-5442 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 242 and a light chain region corresponding to SEQ ID NO: 1.

  TPP-8825 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 243 and a light chain region corresponding to SEQ ID NO: 1.

  TPP-2149 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 12 and a light chain region corresponding to SEQ ID NO: 11.

  TPP-2093 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 22 and a light chain region corresponding to SEQ ID NO: 21.

  TPP-2148 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 32 and a light chain region corresponding to SEQ ID NO: 31.

  TPP-2084 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 42 and a light chain region corresponding to SEQ ID NO: 41.

  TPP-2077 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 52 and a light chain region corresponding to SEQ ID NO: 51.

  TPP-1538 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 62 and a light chain region corresponding to SEQ ID NO: 61.

  TPP-883 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 72 and a light chain region corresponding to SEQ ID NO: 71.

  TPP-1854 is an antibody comprising a heavy chain region sequence corresponding to number 82 and a light chain region corresponding to SEQ ID NO: 81.

  TPP-1853 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 92 and a light chain region corresponding to SEQ ID NO: 91.

  TPP-1857 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 102 and a light chain region corresponding to SEQ ID NO: 101.

  TPP-1858 is an antibody comprising a heavy chain region sequence corresponding to number 112 and a light chain region corresponding to SEQ ID NO: 111.

  TPP-2090 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 10 and a light chain variable region corresponding to SEQ ID NO: 9.

  TPP-2149 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 20 and a light chain variable region corresponding to SEQ ID NO: 19.

  TPP-2093 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 30 and a light chain variable region corresponding to SEQ ID NO: 29.

  TPP-2148 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 40 and a light chain variable region corresponding to SEQ ID NO: 39.

  TPP-2084 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 50 and a light chain variable region corresponding to SEQ ID NO: 49.

  TPP-2077 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 60 and a light chain variable region corresponding to SEQ ID NO: 59.

  TPP-1538 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 70 and a light chain variable region corresponding to SEQ ID NO: 69.

  TPP-883 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 80 and a light chain variable region corresponding to SEQ ID NO: 79.

  TPP-1854 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 90 and a light chain variable region corresponding to SEQ ID NO: 89.

  TPP-1853 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 100 and a light chain variable region corresponding to SEQ ID NO: 99.

  TPP-1857 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 110 and a light chain variable region corresponding to SEQ ID NO: 109.

  TPP-1858 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 120 and a light chain variable region corresponding to SEQ ID NO: 119.

Table: DNA sequences of antibodies according to the invention

  Preferred embodiments of anti-TWEAKR antibodies are:

  1. An anti-TWEAKR antibody, deglycosylated variant, or antigen-binding fragment thereof that binds specifically to D at position 47 (D47) of TWEAKR (SEQ ID NO: 169).

  2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein the antibody is an agonist antibody.

3.
(A) CDR1 of the heavy chain encoded by an amino acid sequence (SEQ ID NO: 171) comprising the formula PYPMX (X is I or M);
(B) the heavy chain CDR2 encoded by the amino acid sequence (SEQ ID NO: 172) comprising the formula YISPSGGXTHYADSVKG (X is S or K); and (c) the amino acid sequence comprising the formula GGDTYFDYFDY (SEQ ID NO: 173) CDR3 of the modified heavy chain
And (a) the CDR1 of the light chain encoded by the amino acid sequence (SEQ ID NO: 174) comprising the formula RASQSISXYLN (X is G or S);
(B) the light chain CDR2 encoded by the amino acid sequence (SEQ ID NO: 175) comprising the formula XASSLQS (X is Q, A or N); and (c) the formula QQSYXPXIT (X at position 5 is T or CDR of the light chain encoded by the amino acid sequence (SEQ ID NO: 176) comprising S, X at position 6 is T or S, and X at position 8 is G or F.
The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a variable light chain comprising

4).
a. A variable heavy chain comprising the heavy chain variable CDR1 sequence shown in SEQ ID NO: 6, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 7 and the heavy chain variable CDR3 sequence shown in SEQ ID NO: 8, and A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 4 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 5, or b. The variable heavy chain comprising the heavy chain variable CDR1 sequence shown in SEQ ID NO: 16, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 17, the heavy chain variable CDR3 sequence shown in SEQ ID NO: 18, and the SEQ ID NO: 13 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 14 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 15; or c. The heavy chain variable CDR1 sequence shown in SEQ ID NO: 26, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 27, the variable heavy chain comprising the heavy chain variable CDR3 sequence shown in SEQ ID NO: 28, and further in SEQ ID NO: 23 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 24 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 25, or d. The heavy chain variable CDR1 sequence shown in SEQ ID NO: 36, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 37, the variable heavy chain comprising the heavy chain variable CDR3 sequence shown in SEQ ID NO: 38, and further in SEQ ID NO: 33 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 34 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 35, or e. The heavy chain variable CDR1 sequence shown in SEQ ID NO: 46, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 47, the variable heavy chain comprising the heavy chain variable CDR3 sequence shown in SEQ ID NO: 48, and further in SEQ ID NO: 43 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 44 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 45, or f. The heavy chain variable CDR1 sequence shown in SEQ ID NO: 56, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 57, the variable heavy chain comprising the heavy chain variable CDR3 sequence shown in SEQ ID NO: 58, and further SEQ ID NO: 53 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 54 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 55, or g. The heavy chain variable CDR1 sequence shown in SEQ ID NO: 66, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 67, the variable heavy chain comprising the heavy chain variable CDR3 sequence shown in SEQ ID NO: 68, and further in SEQ ID NO: 63 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 64, and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 65, or h. The heavy chain variable CDR1 sequence shown in SEQ ID NO: 76, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 77, the variable heavy chain comprising the heavy chain variable CDR3 sequence shown in SEQ ID NO: 78, and further in SEQ ID NO: 73 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 74 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 75, or i. A variable heavy chain comprising the heavy chain variable CDR1 sequence set forth in SEQ ID NO: 86, the heavy chain variable CDR2 sequence set forth in SEQ ID NO: 87, the heavy chain variable CDR3 sequence set forth in SEQ ID NO: 88, and A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 84 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 85, or j. The heavy chain variable CDR1 sequence shown in SEQ ID NO: 96, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 97, the variable heavy chain comprising the heavy chain variable CDR3 sequence shown in SEQ ID NO: 98, and further in SEQ ID NO: 93 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 94 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 95, or k. A variable heavy chain comprising the heavy chain variable CDR1 sequence depicted in SEQ ID NO: 106, a heavy chain variable CDR2 sequence depicted in SEQ ID NO: 107, a heavy chain variable CDR3 sequence depicted in SEQ ID NO: 108, and further comprising SEQ ID NO: 103 A variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 104 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 105 or l. The heavy chain variable CDR1 sequence shown in SEQ ID NO: 116, the heavy chain variable CDR2 sequence shown in SEQ ID NO: 117, the variable heavy chain comprising the heavy chain variable CDR3 sequence shown in SEQ ID NO: 118, and further in SEQ ID NO: 113 According to any of the preceding embodiments comprising a variable light chain comprising the variable CDR1 sequence of the indicated light chain, the variable CDR2 sequence of the light chain set forth in SEQ ID NO: 114 and the variable CDR3 sequence of the light chain set forth in SEQ ID NO: 115 An antibody or antigen-binding fragment thereof.

5.
a. A variable sequence of the heavy chain set forth in SEQ ID NO: 10 and further a variable sequence of the light chain set forth in SEQ ID NO: 9, or b. A heavy chain variable sequence set forth in SEQ ID NO: 20 and a light chain variable sequence set forth in SEQ ID NO: 19, or c. A heavy chain variable sequence set forth in SEQ ID NO: 30 and a light chain variable sequence set forth in SEQ ID NO: 29, or d. A heavy chain variable sequence set forth in SEQ ID NO: 40 and a light chain variable sequence set forth in SEQ ID NO: 39, or e. The heavy chain variability set forth in SEQ ID NO: 50, and further the light chain variability sequence set forth in SEQ ID NO: 49, or f. A heavy chain variable sequence set forth in SEQ ID NO: 60, and a light chain variable sequence set forth in SEQ ID NO: 59, or g. The variable sequence of the heavy chain set forth in SEQ ID NO: 70, and also the variable sequence of the light chain set forth in SEQ ID NO: 69, or h. The heavy chain variable sequence set forth in SEQ ID NO: 80, and also the light chain variable sequence set forth in SEQ ID NO: 79, or i. A heavy chain variable sequence set forth in SEQ ID NO: 90 and a light chain variable sequence set forth in SEQ ID NO: 89, or j. A heavy chain variable sequence set forth in SEQ ID NO: 100, and a light chain variable sequence set forth in SEQ ID NO: 99, or k. The heavy chain variable sequence set forth in SEQ ID NO: 110 and the light chain variable sequence set forth in SEQ ID NO: 109, or l. An antibody or antigen-binding fragment thereof according to any of the previous embodiments, comprising the variable sequence of the heavy chain set forth in SEQ ID NO: 120, and further the variable sequence of the light chain set forth in SEQ ID NO: 119.

  6). The antibody according to any of the previous embodiments which is an IgG antibody.

7).
a. The sequence of the heavy chain set forth in SEQ ID NO: 2 and the sequence of the light chain set forth in SEQ ID NO: 1, or b. The sequence of the heavy chain set forth in SEQ ID NO: 12 and further the sequence of the light chain set forth in SEQ ID NO: 11, or c. The sequence of the heavy chain set forth in SEQ ID NO: 22 and further the sequence of the light chain set forth in SEQ ID NO: 21, or d. The sequence of the heavy chain set forth in SEQ ID NO: 32 and the sequence of the light chain set forth in SEQ ID NO: 31, or e. The sequence of the heavy chain set forth in SEQ ID NO: 42 and the sequence of the light chain set forth in SEQ ID NO: 41, or f. The sequence of the heavy chain set forth in SEQ ID NO: 52 and the sequence of the light chain set forth in SEQ ID NO: 51, or g. The sequence of the heavy chain set forth in SEQ ID NO: 62 and further the sequence of the light chain set forth in SEQ ID NO: 61, or h. The sequence of the heavy chain set forth in SEQ ID NO: 72 and the sequence of the light chain set forth in SEQ ID NO: 71, or i. The sequence of the heavy chain set forth in SEQ ID NO: 82 and the sequence of the light chain set forth in SEQ ID NO: 81, or j. The sequence of the heavy chain set forth in SEQ ID NO: 92, and further the sequence of the light chain set forth in SEQ ID NO: 91, or k. The sequence of the heavy chain set forth in SEQ ID NO: 102 and the sequence of the light chain set forth in SEQ ID NO: 101, or l. An antibody according to any of the previous embodiments comprising the heavy chain sequence set forth in SEQ ID NO: 112 and the light chain sequence set forth in SEQ ID NO: 111.

  8). An antigen-binding fragment according to any of the previous embodiments or an antigen-binding fragment of an antibody according to any of the previous embodiments which is an scFv, Fab, Fab ′ fragment or F (ab ′) 2 fragment.

  9. The antibody or antigen-binding fragment thereof according to any of the previous embodiments, which is a monoclonal antibody or an antigen-binding fragment thereof.

  10. The antibody or antigen-binding fragment thereof according to any of the preceding embodiments which is a human, humanized or chimeric antibody or antigen-binding fragment.

  Particularly preferred is anti-TWEAKR antibody TPP-2090.

  One embodiment of the present invention provides antibodies suitable for transglutaminase (TGase) mediated complexation of kinesin spindle protein inhibitors.

  The human isotype wild-type full-length IgG antibody has a conserved acceptor glutamine at residue 295 (Kabat EU numbering) in the heavy chain that is accessible and reactive in the presence of TGase, so that the antibody is unglycosylated. In some cases, a complex is formed from the antibody and a suitable compound. Such “deglycosylated” or “deglycosylated antibodies” or “deglycosylated antibodies” comprise an Fc region that does not have a glycan attached to a conserved N-linked site in the CH2 domain of the Fc region.

  Deglycosylated antibodies can be generated, for example, by expressing the antibodies in an expression system that does not have glycosylation. Deglycosylated antibodies can be produced by expression of the antibody in a prokaryotic host. Suitable prokaryotic hosts include E. coli, Bacillus subtilis, Salmonella typhimurium and various species of Pseudomonas, Streptomyces and Staphylococcus. It is not limited to. In another embodiment of the invention, deglycosylation antibodies can be achieved using a mammalian expression system with the glycosylation inhibitor tunicamycin (Nose & Wigell (1983), Proc Natl Acad Sci USA, 80 (21). : 6632-6). That is, the modification is prevention of glycosylation at a conserved N-linked site in the CH2 domain of the Fc portion of the antibody. In another embodiment of the invention, glycans bound to conserved N-linked sites in the CH2 domain of Fc region antibodies are removed, which means that the antibody is deglycosylated. Methods for enzymatic deglycosylation of antibodies are known in the art (eg, Winkelhave & Nicolson (1976), J Biol Chem. 251 (4): 1074-80). Deglycosylated antibodies can be produced, for example, by enzymatic deglycosylation using PNGaseF.

  In another embodiment of the invention, deglycosylated antibodies are produced by mutation of the heavy chain glycosylation site of N297 (using Kabat EU numbering). Enzymatic conjugation of such modified deglycosylated antibodies is the mutation N297D, N297Q (Jeger et al., Angelwandte Chemie Int. Ed. Engl 49, 9995-9997 (2010)), or N297S (patent application WO2013092998A1). And WO2013092983A2) have been described. Furthermore, the present invention demonstrates that transglutaminase can efficiently catalyze complexation to a deglycosylated antibody variant with the mutation N297A (Kabat EU numbering).

  Additional sites or other sites reactive in the presence of TGase can be created by engineering the antibody. The compounds of the present invention have one or more amino acids in the wild-type or parent antibody replaced with a glutamine amino acid (substituting thereby), or the glutamine residue suitably comprises another amino acid residue (eg, an acceptor glutamine residue) A glutamine modified antibody introduced or added to the wild type or parent.

  Single site mutations providing glutamine accessible to TGase can result in multiple modified glutamine residues that can be complexed when the antibody contains multiple modified chains. For example, single site mutations result in two modified glutamine residues in IgG due to the dimerization of IgG antibodies.

  The glutamine amino acid residues of an antibody that are reactive in the presence of TGase under suitable conditions can be in the heavy chain, typically in the heavy chain in the constant region. In one embodiment, the asparagine at amino acid position 297 (Kabat EU numbering) is replaced with a residue that is different from glutamine. Preferred is N297D, N297Q, N297S or N297A, and very preferred is N297A. The antibody has a constant region with N297X substitution. Thus, an antibody having an N297X substitution and glutamine at residue 295 (Kabat EU numbering) will have one acceptor glutamine and thus one binding site per heavy chain. Thus, the complete IgG type will have two complexes per antibody.

  The glutamine amino acid residues of an antibody that are reactive in the presence of TGase under suitable conditions can be in the heavy chain, typically in the heavy chain in the constant region. In one embodiment, the asparagine at amino acid position 297 (Kabat EU numbering) is replaced with a glutamine residue. The antibody has a constant region with N297Q substitution. Thus, an antibody having an N297Q substitution and glutamine at residue 295 (Kabat EU numbering) will have two acceptor glutamines and thus two binding sites per heavy chain. Thus, the complete IgG type will have four complexes per antibody.

  The glutamine amino acid residues of an antibody that are reactive in the presence of TGase under suitable conditions can be in the heavy chain, typically in the heavy chain in the constant region. In one embodiment, the asparagine at amino acid position 297 (Kabat EU numbering) is substituted with a glutamine residue, and glutamine is replaced at position 295 (Kabat EU numbering). The antibody will have constant regions with N297Q and Q295X substitutions. Preferred is Q295N substitution. Thus, an antibody with an N297Q substitution and no residue at residue 295 (EU numbering) will have one acceptor glutamine and thus one binding site per heavy chain. Thus, the complete IgG type will have two complexes per antibody.

Preferred antibodies suitable for transglutaminase (TGase) mediated complexation are:
i. N297X substitutions where X is an amino acid other than asparagine (even more preferred are N297D, N297Q, N297S or N297A, very preferred are N297A and N297Q).
ii. N297Q and Q295X substitutions where X is an amino acid other than glutamine (preferably Q295N)
including.

  Thus, an advantageous approach for producing conjugated antibodies involves providing an antibody that does not have N297-linked glycosylation as a source (such N-linked glycosylation interferes with TGase coupling).

Isotopes, salts, solvates, isotopic variants The present invention also encompasses all suitable isotopic forms of the compounds according to the invention. The isotopic forms of the compounds according to the invention are those in which, in this specification, at least one atom in the compounds according to the invention has the same atomic number but has an atomic mass different from the atomic mass that is normally or predominantly in nature. Is understood to mean a compound that has been exchanged for Examples of isotopes that can be incorporated into the compounds according to the invention are the isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, for example ² H (deuterium), ³ H (tritium), ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³² P, ³³ P, ³³ S, ³⁴ S, ³⁵ S, ³⁶ S, ¹⁸ F, ³⁶ Cl, ⁸² Br, ¹²³ I ¹²⁴ I, ¹²⁹ I and ¹³¹ I. Certain isotopic forms of the compounds according to the invention, in particular those incorporating one or more radioactive isotopes, may be useful, for example, for testing the mechanism of action or active compound distribution in the body. In particular, compounds labeled with ³ H or ¹⁴ C isotopes are suitable for that purpose because they are relatively easy to produce and detect. Furthermore, the incorporation of isotopes, such as deuterium, can be particularly therapeutically beneficial by increasing the metabolic stability of the compound, for example, increasing the half-life in the body or the required active ingredient dose. Decrease. Accordingly, such modifications of the compounds according to the invention may optionally constitute preferred embodiments of the invention. The isotopic forms of the compounds according to the invention can be determined by using the corresponding isotopic modifications of the individual reagents and / or starting compounds by methods known to those skilled in the art, for example by the methods described below and the procedures described in the examples. Can be manufactured.

Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. Also included are salts that are not suitable per se for pharmaceutical use, but can be used, for example, for the isolation or purification of the compounds according to the invention.

  Physiologically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzene Examples include sulfonic acid, toluenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid, and benzoic acid salts.

  Physiologically acceptable salts of the compounds of the present invention further include conventional base salts such as, preferably, alkali metal salts (eg, sodium and potassium salts), alkaline earth metal salts (eg, calcium and magnesium salts) and Ammonia or an organic amine having 1 to 16 carbon atoms, for example, preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, And ammonium salts derived from N-methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylenediamine.

Solvates in the context of the present invention are described as the form of the compounds according to the invention forming a solid or liquid complex by coordination with solvent molecules. Hydrates are a specific form of solvates whose coordination is due to water. Preferred solvates in the context of the present invention are hydrates.

  The present invention further encompasses prodrugs of the compounds according to the invention. The term “prodrug” as used herein is a compound that may be physiologically active or inactive, but is converted to a compound of the invention (eg, metabolically or hydrolysed) in the body. Point to.

Specific Embodiments The following embodiments are particularly preferred.

Embodiment A:
ADC of the following formula (or APDC).

[Wherein, KSP-L- represents any of the formulas (II), (IIa), (IIIb), (IIIc), (IIId), (IIIe), (IV) to (IX)), or the following formula (IIf And the binder is an acceptor glutamine residue (particularly preferably an anti-TWEAKR antibody or a variant thereof that specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169) Anti-TWEAKR antibody TPP-2090), deglycosylated variants of these antibodies formed by deglycosylation with PNGaseF or by mutation of heavy chain N297 (Kabat numbering) to either amino acid, and bacterial transglutaminase Modified to include a solvent accessible glutamine residue that is a substrate of An anti-TWEAKR antibody comprising a variant of the antibody,
n is 2 or 4,
Formula (IIf):

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C;
X ₁ represents CH, X ₂ represents C, X ₃ represents N;
X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH, X ₂ represents N, and X ₃ represents C;
A represents C (= O);
R ¹ is —L— # 1, —H, —COOH, —C (═O) —NHNH ₂ , — (CH ₂ ) _1-3 NH ₂ , —C (═O) —NZ ″ (CH ₂ ) _{1 −3 represents} NH ₂ and —C (═O) —NZ ″ CH ₂ COOH, Z ″ represents —H or —NH ₂ ;
R ² and ^{R 4} represents -H or-L-# 1, or ^{R 2} represents -H, ^{R 4} is _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2 ₎ —P ₂ —NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) — (P 2 and Pe have the same meaning as defined above, eg as indicated for formula (IIa) . with) it represents, or ^{R 2} and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - ^{represents, R 10} is -H or -L- Represents # 1;
R ³ represents —L— # 1 or C _1-10 -alkyl-, which represents —OH, —O-alkyl, —SH, —S-alkyl, —O—C (O) -alkyl, —O—C. (═O) —NH-alkyl, —NH—C (═O) -alkyl, —NH—C (═O) —NH-alkyl, —S (═O) _n -alkyl, —S (═O) ₂ Optionally substituted by -NH-alkyl, -NH-alkyl, -N (alkyl) ₂ or -NH ₂ (alkyl is preferably C _1-3 -alkyl);
R ⁵ represents -L- # 1, H or F;
R ⁶ and R ⁷ are independently of each other —H, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _2-10 —. Represents alkynyl, fluoro- _C2-10 -alkynyl, hydroxy or halogen (especially -F, -Cl, -Br);
R ⁸ represents a branched C _1-5 -alkyl group, which may be substituted by -L- # 1;
R ⁹ represents H or F;
One of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents -L- # 1;
-L- represents a linker and # 1 represents binding to the antibody. ]
And its ADC salts, solvates and solvate salts.

The linker is preferably the following linker.

Where
m is 0 or 1;
§ represents binding to KSP,
§§ represents antibody binding,
L2 represents # ^1- (NH) _p- (C = O) _q- G4-NH- # ² or # ^1- (NH) _p- (C = O) _q- G4-O-NH- # ^2.
Represents
p is 0 or 1;
q is 0 or 1;
G4 represents an optionally substituted alkyl or heteroalkyl chain, where any carbon of the chain is alkoxy, hydroxyl, alkylcarbonyloxy, -S-alkyl, thiol, -C (= O) -S - alkyl, -C (= O) -NH- alkyl, -NH-C (= O) - alkyl, amine is substituted with -C (= O) -NH _2,
# ¹ represents the point of attachment to the group ^{L 1,}
# ² indicates the binding site of the binder to the glutamine residue,
L1 is the following formula:

Represented by
Where
R ¹⁰ represents —H, —NH ₂ or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) — or

Represents;
n is 0 or 1;
o is 0 or 1;
G3 represents an optionally substituted straight or branched hydrocarbon chain having 1 to 100 carbon atoms from a bond or arylene group and / or straight chain and / or branched and / or cyclic alkylene group, Groups -O-, -S-, -S (= O)-, -S (= O) ₂ , -NH-, -C (= O)-, -NH-C (= O)-, -C ( = O) -NH-, -NMe-, -NHNH-, -S (= O) _2- NHNH-, -C (= O) -NHNH- and N, O and S or -S (= O)-( Preferably

Or

) May be interrupted one or more times by one or more of 3 to 10 membered aromatic or non-aromatic heterocycles having 4 or fewer heteroatoms selected from the group consisting of: when present, it _is -NH-C (= O) -NH 2, -COOH, -OH, -NH 2, -NH-CNNH 2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid.

Here, # ¹ is a bond to a KSP inhibitor, and # ² is a bond to a coupling group to a binder (eg, L2).

Embodiment B:
ADC of the following formula:

[Where:
KSP-L- may be any one of formulas (II), (IIa), (IIIb), (IIIc), (IIId), (IIIe), (IIf), (IV) to (IX) or the following formula ( IIg), the binder is an antibody containing an aquaceptor glutamine residue, n is 2 or 4,
Formula (IIg):

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C;
X ₁ represents CH, X ₂ represents C, X ₃ represents N;
X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH, X ₂ represents N, and X ₃ represents C;
A represents -C (= O)-;
R ¹ is —L— # 1, —H, —COOH, —C (═O) —NHNH ₂ , — (CH ₂ ) _1-3 NH ₂ , —C (═O) —NZ ″ (CH ₂ ) _{1 −3 represents} NH ₂ and —C (═O) —NZ ″ CH ₂ COOH, Z ″ represents —H or —NH ₂ ;
R ² and R ⁴ represent —H or —L- # 1, or R ² represents —H, and R ⁴ represents R ²¹ — (C═O) ( _0-1) — (P3) _{(0— 2) -P2-NH-CH (} CH 2 C (= O) -NH 2) -C (= O) - (P2 and Pe, for example formula (IIa) as defined hereinabove above as indicated with respect to . meaning having a) represents, or ^{R 2} and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - ^{represents, R 10} is -H or -L -Represents # 1;
R ³ represents —L— # 1 or C _1-10 -alkyl-, which represents —OH, —O-alkyl, —SH, —S-alkyl, —O—C (═O) -alkyl, —O—. C (= O) -NH-alkyl, -NH-C (= O) -alkyl, -NH-C (= O) -NH-alkyl, -S (= O) _n -alkyl, -S (= O) ₂ -NH- alkyl, -NH- alkyl, -N _(alkyl) may be substituted by ₂ or -NH ₂ -; (alkyl is _{preferably, C 1-3} alkyl.)
R ⁵ represents -L- # 1, H or F;
R ⁶ and R ⁷ are independently of each other —H, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _2-10 —. Represents alkynyl, fluoro- _C2-10 -alkynyl, hydroxy or halogen (especially -F, -Cl, -Br);
R ⁸ represents a branched C _1-5 -alkyl group;
R ⁹ represents H or F;
One of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents -L- # 1;
-L- represents a linker, # 1 represents binding to the antibody,
-L- is

Represented by
Where
m is 0 or 1;
§ represents binding to KSP,
§§ represents antibody binding,
L2 represents # ^1- (NH) _p- (C = O) _q- G4-NH- # ² or # ^1- (NH) _p- (C = O) _q- G4-O-NH- # ^2.
Represents
p is 0 or 1;
q is 0 or 1;
G4 represents an optionally substituted alkyl or heteroalkyl chain, where suitably any carbon of the chain is alkoxy, hydroxyl, alkylcarbonyloxy, -S-alkyl, thiol, -C (= O) -S - alkyl, -C (= O) -NH- alkyl, -NH-C (= O) - alkyl, amine is substituted with -C (= O) -NH _2,
# ¹ represents the point of attachment to the group ^{L 1,}
# ² indicates the binding site to the glutamine residue of the binder,
L1 is the following formula:

Represented by
R ¹⁰ represents —H, —NH ₂ or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) — or

Represents
n is 0 or 1;
o is 0 or 1;
G3 represents an optionally substituted straight or branched hydrocarbon chain having 1 to 100 carbon atoms from a bond or arylene group and / or straight chain and / or branched and / or cyclic alkylene group, Groups -O-, -S-, -S (= O)-,
—S (═O) ₂ , —NH—, —C (═O) —, —NH—C (═O) —, —C (═O) —NH—, —NMe—, —NHNH—, —S (= O) _2- NHNH-, -C (= O) -NHNH-, and a 3 to 10 membered aromatic or non-aromatic heterocycle N, O having 4 or fewer heteroatoms selected from the group consisting of And S or -S (= O)-(preferably

Or

) May be interrupted one or more times by one or more of the following, and when a side chain is present, it is —NH—C (═O) —NH ₂ , —COOH, —OH, —NH ₂ , — NH-CNNH _2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid,
# ¹ is the bond to the KSP inhibitor and # ² is the bond to the coupling group for the antibody (eg L2). ]
And its ADC salts, solvates and solvate salts.

Embodiment C:
ADC of the following formula:

[Wherein KSP-L- is a compound of the following substructure I (sub) and the binder is an acceptor glutamine residue (particularly preferably at position 47 (D47) of trastuzumab or TWEAKR (SEQ ID NO: 169)). Anti-TWEAKR antibody that specifically binds to amino acid D, especially anti-TWEAKR antibody TPP-2090), anti-HER2 antibody or anti-EGRF antibody (preferably nimotuzumab), deglycosylation with PNGaseF or N297 of heavy chain (Kabat numbering) Deglycosylated variants of these antibodies formed by mutation to any of the amino acids, and the aforementioned modified to include one or more acceptor glutamine residues for transglutaminase (TGase) catalysis Antibody variant, n is 2 The other is 4;

Where
#A represents binding to the rest of the molecule;
R ^1a represents —L— # 1, —H or — (CH ₂ ) _0-3 Z, Z represents —H, halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C ( = O) -OY ³
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH,
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
R ^2a and R ^4a each independently represent -L- # 1, H, -C (= O) -CHY ⁴ -NHY ⁵ or-(CH ₂ ) _0-3 Z;
Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ^2a represents -H;
R ^4a is R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) — Represents
P2 and P3 have the same meaning as defined above, for example as indicated for formula (IIa)
R ²¹ is H, C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6- 10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl, - N _{(alkyl) 2, -NH-C (=} O) - alkyl, N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -S (=} O) 2 NH 2, -S (= O ) ₂ -N (A _{Kill) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _{alkyl) 2} or may optionally be substituted one or more times by -OH,, or, -H or a group - _(O) x - represents a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
Or R ^2a and ^{R 4a} together form (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents _{-H, -L- # 1, -NH 2} , -SO 3 H, -COOH, -SH, and -SO ₃ H or -OH,
One of the substituents R ^1a , R ^2a , R ^4a or R ¹⁰ represents -L- # 1;
-L- represents a linker, # 1 represents binding to the antibody,
-L- is

Represented by;
Where
m is 0 or 1;
§ represents binding to KSP,
§§ represents antibody binding,
L2

Represents
p is 0 or 1;
q is 0 or 1;
G4 represents an optionally substituted alkyl or heteroalkyl chain, where suitably any carbon of the chain is alkoxy, hydroxyl, alkylcarbonyloxy, -S-alkyl, thiol, -C (= O) -S - alkyl, -C (= O) -NH- alkyl, -NH-C (= O) - alkyl, amine is substituted with -C (= O) -NH _2,
# ¹ represents the point of attachment to the group ^{L 1,}
# ² indicates the binding site of the binder to the glutamine residue,
L1 is the following formula:

Represented by;
Where
R ¹⁰ represents —H, —NH ₂ or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) — or

Represents;
n is 0 or 1;
o is 0 or 1;
G3 represents an optionally substituted straight or branched hydrocarbon chain having 1 to 100 carbon atoms from a bond or arylene group and / or straight chain and / or branched and / or cyclic alkylene group, Groups -O-, -S-, -S (= O)-, -S (= O) ₂ , -NH-, -C (= O)-, -NH-C (= O)-, -C ( ═O) —NH—, —NMe—, —NHNH—, —S (═O) ₂ —NHNH—, —C (═O) —NHNH— and N, O and S or —S (═O) — A 3- to 10-membered aromatic or non-aromatic heterocycle having 4 or fewer heteroatoms selected from the group consisting of (preferably

Or

) May be interrupted one or more times by one or more of the following, and when a side chain is present, it is —NH—C (═O) —NH ₂ , —COOH, —OH, —NH ₂ , — NH-CN-NH _2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid.

Here, # ¹ is a bond to a KSP inhibitor, and # ² is a bond to a coupling group to a binder (eg, L2). ]
And its ADC salts, solvates and solvate salts.

Embodiment D:
ADC of the following formula:

[Wherein KSP-L- represents a compound represented by the formula (II), (IIa), (IIIb), (IIIc), (IIId), (IIe), (IIf), (IIg), (III) to (IX) Or a compound of formula (IIh) below, wherein the binder is an antibody comprising an aquaceptor glutamine residue and n is a number 2 or 4;

Where
X ₁ represents N, X ₂ represents N, X ₃ represents C;
X ₁ represents CH, X ₂ represents C, X ₃ represents N;
X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH, X ₂ represents N, and X ₃ represents C;
A represents -C (= O)-;
R ¹ represents -L- # 1;
R ² and R ⁴ represent —H,
Or R ² represents -H;
R ⁴ is _{^{R 21 -C (= O) -P3}} (0-2) -P2-NH-CH (CH 2 C (= O) -NH 2) -C (= O) - represents,
P2 and P3 have the same meaning as defined above, for example as indicated for formula (IIa)
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - ^{represents, R 10} represents H;
R ³ represents C _1-10 -alkyl, which is represented by 1 to 3 —OH groups, 1 to 3 —O-alkyl groups, 1 to 3 —SH groups, 1 to 3 —S—. An alkyl group, 1 to 3 —O—C (═O) -alkyl groups, 1 to 3 —O—C (═O) —NH-alkyl groups, 1 to 3 —NH—C (═ O) -alkyl group, 1 to 3 —NH—C (═O) —NH-alkyl group, 1 to 3 —S (═O) _n -alkyl group, 1 to 3 —S (═ O) ₂ -NH-alkyl, optionally substituted by 1 to 3 -NH-alkyl, 1 to 3 -N (alkyl) ₂ or 1 to 3 -NH ₂ ;
Alkyl is preferably C _1-3 -alkyl or -MOD,
-MOD represents-(NR _< ¹⁰ _> ) _n- (G1) _o -G2-H,
R ¹⁰ represents —H or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is —NH—C (═O) — or

R ¹⁰ does not represent NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , -C (= O) -NR ^y NR ^y- may be interrupted one or more times by one or more,
R ^y represents —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NH—C (═O) —NH ₂ , — _{COOH, -OH, -NH 2, NH} -CN-NH 2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid, or ^{-C (= O) -, -} CR x = N-O- Represents
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl;
Hydrocarbon chain containing either side _{chains, -NH-C (= O)} -NH 2, -COOH, -OH, -NH 2, NH-CN-NH 2, sulfonamide, sulfone, sulfoxide or sulfone acid May be replaced by
-MOD preferably has at least one group -COOH;
R ⁵ represents H or F;
R ⁶ and R ⁷ are independently of each other —H, C _1-3 -alkyl, fluoro-C _1-3 -alkyl, C _2-4 -alkenyl, fluoro-C _2-4 -alkenyl, C _2-4- Represents alkynyl, fluoro- _C2-4 -alkynyl, hydroxy or halogen (especially -F, -Cl, -Br);
R ⁸ represents a branched C _1-5 -alkyl group;
R ⁹ represents H or F;
-L- represents a linker, # 1 represents binding to the antibody,
-L- is

Represented by
m is 0 or 1;
§ represents binding to KSP,
§§ represents antibody binding,
L2

Represents
Where
p is 0 or 1;
q is 0 or 1;
G4 represents an optionally substituted alkyl or heteroalkyl chain, where suitably any carbon of the chain is alkoxy, hydroxyl, alkylcarbonyloxy, -S-alkyl, thiol, -C (= O) -S - alkyl, -C (= O) -NH- alkyl, -NH-C (= O) - alkyl, amine, -C (= O) # ¹ is substituted by -NH ₂ is to group ^{L 1} Show the joint location,
# ² indicates the binding site of the binder to the glutamine residue,
L1 is the following formula:

Represented by;
Where
R ¹⁰ represents —H, —NH ₂ or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) — or

Represents;
n is 0 or 1;
o is 0 or 1;
G3 represents an optionally substituted straight or branched hydrocarbon chain having 1 to 100 carbon atoms from a bond or arylene group and / or straight chain and / or branched and / or cyclic alkylene group, Groups -O-, -S-, -S (= O)-,
—S (═O) ₂ , —NH—, —C (═O) —, —NH—C (═O) —, —C (═O) —NH—, —NMe—, —NHNH—, —S 3 to 10 having 4 or fewer heteroatoms selected from the group consisting of (═O) ₂ —NHNH—, —C (═O) —NHNH— and N, O and S or —S (═O) —. Membered aromatic or non-aromatic heterocycles (preferably

Or

)) May be interrupted one or more times by one or more of the following, and when a hydrocarbon chain containing a side chain is present, it is —NH—C (═O) —NH ₂ , —COOH, —OH, -NH _2, -NH-CNNH _2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid,
# ¹ is the bond to the KSP inhibitor and # ² is the bond to the coupling group for the antibody (eg L2). ]
And salts, solvates, solvate salts and epimers of ADC.

Embodiment E:
Site-specific and homogeneous ADC of the formula:

  Wherein KSP-L- is a compound of the formula (II), (IIa), (IIIb), (IIIc), (IIId), (IIe), (IIf), (IIg), or (III) to (IX) Or a compound of formula (IIh). And ADC salts, solvates and solvate salts.

One embodiment of the present invention is a kinesin spindle protein inhibitor that is a complex of a binder or derivative thereof with one or more active compound molecules, wherein the active compound molecule is bound to the binder via a linker L. The linker L is bound to the glutamine side chain of the binder, 1 to 5 kinesin spindle protein inhibitors are bound to the linker L, and the kinesin spindle protein inhibitor is represented by the following formula (IIa):

[Where:
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or CF X ₂ represents C and X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH and X ₂ represents N and X ₃ represents C (preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N);
R ¹ is -H, -MOD, -L- # 1 or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ² is -L- # 1, H, -MOD, -C (= O) -CHY 4 -NHY 5 or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents —L— # 1, —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
Z is ^{-H, -OY} 3, -SY 3, represents -NHY 3, -C (= O) -NY 1 Y 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ is _L-# 1, represents _{-H, -NH 2, -SO 3 H} , -COOH, a -SH or -OH,
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is -L- # 1, -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably -L- # 1 or C _1-10 -alkyl , C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl Group, 1 to 3 —SH group, 1 to 3 —S-alkyl group, 1 to 3 —O—C (═O) -alkyl group, 1 to 3 —O—C (═ O) -NH-alkyl group, Of three -NH-C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - group 1 to 3 —S (═O) ₂ —NH-alkyl groups, 1 to 3 —NH-alkyl groups, 1 to 3 —N (alkyl) ₂ groups, 1 to 3 —NH _Optionally substituted by ₂ or 1 to 3 — (CH ₂ ) _0-3 Z groups,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
n represents 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NHC (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) _0-3 represents Z '
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁵ is _-H, -NH _2, -NO _2, halogen (especially -F, -Cl, -Br), - SH or - _{(CH 2)} represents a _0-3 Z,
Z represents -H, -OY ³ , -SY ³ , halogen, -NHY ³ , -C (O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} - represents alkynyl, _hydroxy, -NO _2, -NH 2, -COOH, or halogen (especially -F, -Cl, -Br), and - alkynyl, fluoro _{-C 2-10}
R ⁸ is C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _2-10 -alkynyl, fluoro-C _2-10 -alkynyl , C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl or — (CH ₂ ) _0-2- (HZ ² ),
HZ ² represents a 4 to 7 membered heterocyclic ring (preferably oxetane) having 2 or less heteroatoms selected from the group consisting of N, O and S, each of these groups being —OH, —COOH, It may be substituted by -NH ₂ or-L-# 1;
One or zero of the substituents R ¹ , R ² , R ³ , R ⁴ R ⁵ , R ⁸ and R ¹⁰ represents -L- # 1, or (in the case of R ⁸ ) comprises-
L represents a linker;
# 1 represents a bond to a binder or derivative thereof;
-MOD represents-(NR _< ¹⁰ _> ) _n- (G1) _o -G2-H,
R ¹⁰ represents —H or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is —NH—C (═O) — or

R ¹⁰ does not represent NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y — may be interrupted one or more times by one or more of:
R ^y represents —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NH—C (═O) —NH ₂ , — _{COOH, -OH, -NH 2, NH} -CN-NH 2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid, or ^{-C (= O) -, -} CR x = N-O- Represents
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl;
Hydrocarbon chain containing either side _{chains, -NH-C (= O)} -NH 2, -COOH, -OH, -NH 2, NH-CN-NH 2, sulfonamide, sulfone, sulfoxide or sulfone acid May be replaced by
G3 represents -H or -COOH;
-MOD preferably has at least one group -COOH. ]
As well as salts, solvates, solvate salts and epimers thereof.

Another embodiment of the present invention is the above complex, wherein X ₁ represents CH, X ₂ represents C, and X ₃ represents N.

Another embodiment of the present invention is the above conjugate, wherein the substituent R ¹ represents -L- # 1.

Another embodiment of the invention is a complex with an active compound molecule that is a kinesin spindle protein inhibitor linked to a binder via a linker L of a binder or derivative thereof, wherein the linker L is a glutamine of the binder The kinesin spindle protein inhibitor bound to the side chain has the following substructure:

[Where:
#A represents binding to the rest of the molecule;
R ^1a represents H or — (CH ₂ ) _0-3 Z;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents a linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , and represents aryl or benzyl which may be substituted by —NH ₂ ;
R ^2a and R ^4a each independently represent —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents —H, —NH ₂ , —SO ₃ H, —COOH, —SH, or —OH;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
The kinesin spindle protein inhibitor is attached to the linker by substitution of a hydrogen atom at R ^1a , R ^2a , R ^4a or at the pyrrolidine ring formed by R ^2a and R ^4a . ]
As well as salts, solvates, solvate salts and epimers thereof.

In another embodiment of the present invention, the kinesin spindle protein inhibitor is represented by the following general formula (I):

[Where:
R ^1a represents —H or — (CH ₂ ) _0-3 Z;
Z is ^{-H, -NHY 3, -OY 3,} -SY 3, represents halogen, ^{-C (= O) -NY 1 Y} 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ or —CH (CH ₂ W) Z ′. ,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ^2a and R ^4a each independently represent —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Or R ^2a and ^{R 4a} together form (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents —H, —NH ₂ , —SO ₃ H, —COOH, —SH, or —OH;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ^3a is an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably -L-binder, or C _1-10 -alkyl, C _6-10 -aryl or C _{Represents a 6-10} -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, which is represented by 1 to 3 —OH A group, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl groups, 1 to 3 —SH 1 to 3 —S-alkyl groups, 1 to 3 —O—C (═O) -alkyl groups, 1 to 3 —O—C (═O) —NH-alkyl groups, To -N -C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, the one to three -S (= _{O) n} - alkyl group, one to three —S (═O) ₂ —NH-alkyl, 1 to 3 —NH-alkyl, 1 to 3 —N (alkyl) ₂ , 1 to 3 —NH ₂ or 1 to 3 number of - _{(CH 2) 0-3} may be substituted by the Z group,
n is 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ is _{-H, - (CH 2) 0-3} -CH (NH-C (= O) -CH 3) Z ', - (CH 2) 0-3 -CH (NH 2) Z' or - ( CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
“Alkyl” preferably represents C _1-10 -alkyl);
R ^8a represents C _1-10 -alkyl;
HZ represents a monocyclic or bicyclic heterocycle, which is one or more selected from the group consisting of halogen, C _1-10 -alkyl group, C _6-10 -aryl group and C _6-10 -aralkyl group Which may be substituted by a substituent, which may be substituted by a halogen;
The kinesin spindle protein inhibitor is linked to the linker by hydrogen atom substitution at R ^1a , R ^2a , R ^3a , R ^4a , R ^8a or at the pyrrolidine ring formed by R ^2a and R ^4a . ]
And the salts, solvates and solvate salts thereof as described above.

Another embodiment of the present invention is that the linker L is bound to the glutamine side chain of the binder, 1 to 5 kinesin spindle protein inhibitors are bound to the linker L, and the active compound molecular linker is General formula (II):

[Where:
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH X ₂ represents C and X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH and X ₂ represents N X ₃ represents C;
R ¹ represents -H, -L- # 1 or-(CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ² and R ⁴ each independently represent —L— # 1, —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ² represents -H;
R ⁴ represents R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) -Represents a group of
R ²¹ is H, C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6- 10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl, - N _{(alkyl) 2, -NH-C (=} O) - alkyl, N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -S (=} O) 2 NH 2, -S (= O ) ₂ -N (A _Kill) 2, -COOH,
It may be substituted one or more times with —C (═O) —NH ₂ , —C (═O) —N (alkyl) ₂ , or —OH, or —H or a group — (O) _x — (CH It represents ^{_{2 CH 2 O) y -R 22}} ,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 and P3 have the same meaning as defined above, for example as indicated for formula (IIa)
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ is _L-# 1, represents _{-H, -NH 2, -SO 3 H} , -COOH, a -SH or -OH,
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is -L- # 1 or an optionally substituted alkyl, aryl, heteroaryl, or heterocyclic alkyl group, preferably -L- # 1 or C _1-10 -alkyl, C _6-10 -aryl, _{Represents a} C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, which represents 1 to 3 — OH group, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl groups, 1 to 3 — SH group, 1 to 3 -S-alkyl group, 1 to 3 -O-C (= O) -alkyl group, 1 to 3 -O-C (= O) -NH-alkyl group, 1 to 3 —NH—C (═O) -alkyl , One to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - alkyl group, one to three -S ₍₌ O) 2 -NH -Alkyl group, 1 to 3 -NH-alkyl group, 1 to 3 -N (alkyl) ₂ group, 1 to 3 -NH ₂ group or 1 to 3-(CH ₂ ) _{0- 3} optionally substituted by a Z group,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
n represents 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NHC (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) _0-3 represents Z '
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁵ is _{-L- # 1, -H, -F,} -NH 2, -NO 2, halogen (especially -F, -Cl, -Br), - SH or - a _{(CH 2) 0-3} Z Represent,
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
One substituent of R ¹ , R ² , R ³ , R ⁴ and R ⁵ represents -L- # 1;
L represents a linker, # 1 represents a bond to a binder or a derivative thereof,
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} -alkynyl, fluoro- _C2-10 -alkynyl, hydroxy or halogen (especially -F, -Cl, -Br);
R ⁸ represents C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl, or optionally substituted oxetane;
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ . ]
And the complexes defined above as well as salts, solvates, solvate salts and epimers thereof.

Another embodiment of the present invention is:
R ¹ represents -H, -L- # 1 or-(CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or a derivative thereof,
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ or —CH (CH ₂ W) Z ′. ,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—CHY ⁴ ) _{1− 3} represents COOH,
W represents —H or —OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ Representation;
R ² and R ⁴ independently of each other represent —L— # 1, —H or —C (═O) —CHY ⁴ —NHY ⁵ ;
Or R ² and R ⁴ together (form a pyrrolidine ring) represent —CH ₂ —CHR ¹⁰ —, R ¹⁰ is —H, —L— # 1, —NH ₂ , —COOH, —SH, —OH or an -SO ₃ H,
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ Represent,
Y ⁵ is -H or -C (= O) represents the -CHY ⁶ -NH _2,
Y ⁶ represents linear or branched C _1-6 -alkyl,
A represents -C (= O)-,
R ³ represents — (CH ₂ ) OH or —L- # 1;
R ⁵ represents -L- # 1 or -H;
One of the substituents R ¹ , R ² , R ³ , R ⁴ and R ⁵ represents -L- # 1. ]
And the complexes, salts, solvates, solvate salts and epimers thereof represented by

Another embodiment of the present invention is a complex as defined above, wherein R ⁶ and R ⁷ independently of one another represent —H, C _1-3 -alkyl or halogen.

Another embodiment of the present invention is a complex as defined above wherein R ⁸ represents C _1-4 -alkyl (preferably tert-butyl).

Another embodiment of the present invention is the above defined complex, wherein R ⁹ represents —H.

Another embodiment of the invention is a complex as defined above, wherein R ⁶ and R ⁷ represent —F.

  Another embodiment of the present invention is a complex as defined above, wherein said binder or derivative thereof is a binder peptide or protein or a derivative of a binder peptide or protein.

  Another embodiment of the present invention is a complex as defined above, wherein said complex has two binding sites per binder.

  Another embodiment of the present invention is a complex as defined above, wherein said complex has four binding sites per binder.

  Another embodiment of the present invention is a complex as defined above, wherein said binder peptide or protein represents an antibody or derivative of a binder peptide or protein comprising an acceptor glutamine side chain that can be recognized by transglutaminase.

  Another embodiment of the invention is a complex as defined above produced by transglutaminase mediated complexation.

  Another embodiment of the present invention is a complex as defined above, produced using transglutaminase from Streptomyces Mobaraensis.

  Another embodiment of the present invention is a complex as defined above, wherein said binder binds to a cancer target molecule.

  Another embodiment of the present invention is a complex as defined above, wherein said binder binds to an extracellular target molecule.

  Another embodiment of the invention is as defined above wherein the binder is internalized and processed intracellularly (preferably lysosomally) by a cell expressing the target molecule after binding to the extracellular target molecule. It is a complex.

  Another embodiment of the present invention is a complex as defined above, wherein said binder peptide or protein is a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof.

  Another embodiment of the present invention is the complex as defined above, wherein said binder peptide or protein is an antibody having an acceptor glutamine residue, optionally in the heavy chain in the CH2 domain.

  Another embodiment of the present invention is the complex as defined above, wherein said binder peptide or protein is an antibody having an acceptor glutamine residue in the heavy chain at position 295 (Kabat numbering system).

  Another embodiment of the present invention is that the binder peptide or protein has an N297X substitution (where X is an amino acid other than asparagine), even more preferably N297D, N297Q, N297S or N297A, very preferably N297A and N297Q. It is a complex as defined above which is an antibody comprising.

  Another embodiment of the present invention is the complex as defined above, wherein said binder peptide or protein is an antibody comprising an N297Q substitution and a Q295X substitution (where X is an amino acid other than glutamine), preferably Q295N.

  Another embodiment of the invention is a conjugate as defined above, wherein the binder peptide or protein is an antibody comprising asparagine at residue 297 that has substantially no N-linked glycosylation.

  Another embodiment of the present invention is a conjugate as defined above, wherein said binder peptide or protein is an antibody produced in a host cell that produces an antibody with amino acid residue N297 and no N-linked glycosylation.

  Another embodiment of the present invention is a complex as defined above, wherein said binder peptide or protein is an anti-HER2 antibody, an anti-EGFR antibody, an anti-TWEAKR antibody or an antigen-binding fragment thereof.

  Another embodiment of the present invention is that the anti-TWEAKR antibody specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169), preferably anti-TWEAKR antibody TPP2090 and its deglycosylation mutations A complex as defined above which is a body.

  Another embodiment of the present invention is that the anti-TWEAKR antibody specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169), preferably the anti-TWEAKR antibody TPP-2090-HC-N297A or TPP-2090-HC-N297Q is a complex as defined above.

In another embodiment of the present invention, the linker L is bound to the glutamine side chain of the binder, 1 to 5 kinesin spindle protein inhibitors are bound to the linker L, and the linker L has the following basic structure: (I) to (iv):
(I)-(C = O) _m -SG1-L1-L2-
(Ii)-(C = O) _m -L1-SG-L1-L2-
(Iii)-(C = O) _m -L1-L2-
(Iv)-(C = O) _m -L1-SG-L2
One of
m is 0 or 1, SG and SG1 are in vivo cleavable groups, L1 represents an in vivo and non-cleavable organic group independently of each other, and L2 represents a coupling group to a binder. A complex as defined above.

  In another embodiment of the present invention, the linker L is bound to the glutamine side chain of the binder, 1 to 5 kinesin spindle protein inhibitors are bound to the linker L, and the in vivo cleavable group SG Is a complex as defined above wherein is a 2 to 8 oligopeptide group, preferably a dipeptide group or disulfide, hydrazone, acetal or aminal, and SG1 is a 2 to 8 oligopeptide group, preferably a dipeptide group.

Another embodiment of the invention is a complex as defined above. The linker is attached to the glutamine side chain and has the following formula:

Where
m is 0 or 1;
§ represents binding to the active compound molecule,
§§ represents binding to a binder peptide or protein,
L1 is ^{_{- (NR 10) n - (}} G1) represents o -G3-,
R ¹⁰ represents —H, —H ₂ or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) — or

Represents;
n is 0 or 1;
o is 0 or 1;
G3 represents an optionally substituted straight or branched hydrocarbon chain having 1 to 100 carbon atoms from a bond or arylene group and / or straight chain and / or branched and / or cyclic alkylene group, Groups -O-, -S-, -S (= O)-, -S (= O) ₂ , -NH-, -C (= O)-, -NH-C (= O)-, -C ( ═O) —NH—, —NMe—, —NHNH—, —S (═O) ₂ —NHNH—, —C (═O) —NHNH— and N, O and S or —S (═O) — A 3- to 10-membered aromatic or non-aromatic heterocycle having 4 or fewer heteroatoms selected from the group consisting of (preferably

Or

)) May be interrupted one or more times by one or more of the following, and when a hydrocarbon chain containing a side chain is present, it is —NH—C (═O) —NH ₂ , —COOH, —OH, _{-NH 2, -NH-CN-NH} 2, sulfonamide, sulfone, may be substituted by a sulfoxide or sulfonic acid,
Or the following group:

Represents one of the
Rx is _-H, C 1 -C ₃ - alkyl or phenyl,
L2 represents # ^1- (NH) _p- (C = O) _q- G4-NH- # ² or # ^1- (NH) _p- (C = O) _q- G4-O-NH- # ² ,
p is 0 or 1;
q is 0 or 1;
G4 represents an optionally substituted alkyl or heteroalkyl chain, where suitably any carbon of the chain is alkoxy, hydroxyl, alkylcarbonyloxy, -S-alkyl, thiol, -C (= O) -S - alkyl, -C (= O) -NH- alkyl, -NH-C (= O) - alkyl, amine is substituted with -C (= O) -NH _2,
# ¹ represents the point of attachment to the group ^{L 1,}
# ² represents the binding site to the glutamine residue of the binder.

Another embodiment of the present invention is a complex as defined above, wherein said hydrocarbon chain is interrupted by one of the following groups:

In which X represents —H or a C _1-10 -alkyl group, which represents —NH—C (═O) —NH ₂ , —COOH, —OH, —NH ₂ , —NH—CN—NH ₂ , sulfone. , May be substituted by sulfoxide or sulfonic acid.

Another embodiment of the invention is a complex as defined above. L2 is one of the following groups;
L2 is

(R ^y is —H, —C (═O) —NH-alkyl, —NH—C (═O) -alkyl, —C (═O) —NH ₂ , or —NH ₂ ).
# ¹ represents the point of attachment to the group ^{L 1,}
# ² represents the binding site to the glutamine residue of the binder.

Another embodiment of the present invention is the complex as defined above, wherein R ^y is H or NHCOMe.

Another embodiment of the present invention is the complex as defined above, wherein R ¹ or R ⁴ represents -L- # 1.

  Another embodiment of the present invention is a complex as defined above, wherein said anti-TWEAKR antibody is an agonist antibody.

Another embodiment of the present invention is:
a. CDR1 of the heavy chain encoded by the amino acid sequence comprising the formula PYPMX (SEQ ID NO: 171), where X is I or M;
b. CDR2 of the heavy chain encoded by the amino acid sequence comprising the formula YISPSGGXTHYADSVKG (SEQ ID NO: 172), where X is S or K; and c. CDR3 of the heavy chain encoded by the amino acid sequence comprising the formula GGDTYFDYFDY (SEQ ID NO: 173);
Variable heavy chain containing:
And d. CDR1 of the light chain encoded by the amino acid sequence comprising the formula RASQSISXYLN (SEQ ID NO: 174), where X is G or S;
e. A light chain CDR2 encoded by an amino acid sequence comprising the formula XASSLQS (SEQ ID NO: 175), wherein X is Q, A or N; and f. By an amino acid sequence comprising the formula QQSYXXPXIT (SEQ ID NO: 176) (X at position 5 is T or S, X at position 6 is T or S, and X at position 8 is G or F). CDR3 of the encoded light chain;
A complex as described above comprising a variable light chain comprising

Another embodiment of the present invention is:
a. A variable sequence of the heavy chain set forth in SEQ ID NO: 10 and further a variable sequence of the light chain set forth in SEQ ID NO: 9, or b. A heavy chain variable sequence set forth in SEQ ID NO: 20 and a light chain variable sequence set forth in SEQ ID NO: 19, or c. A heavy chain variable sequence set forth in SEQ ID NO: 30 and a light chain variable sequence set forth in SEQ ID NO: 29, or d. A heavy chain variable sequence set forth in SEQ ID NO: 40 and a light chain variable sequence set forth in SEQ ID NO: 39, or e. A heavy chain variable sequence set forth in SEQ ID NO: 50, and a light chain variable sequence set forth in SEQ ID NO: 49, or f. A heavy chain variable sequence set forth in SEQ ID NO: 60, and a light chain variable sequence set forth in SEQ ID NO: 59, or g. The variable sequence of the heavy chain set forth in SEQ ID NO: 70, and also the variable sequence of the light chain set forth in SEQ ID NO: 69, or h. The heavy chain variable sequence set forth in SEQ ID NO: 80, and also the light chain variable sequence set forth in SEQ ID NO: 79, or i. A heavy chain variable sequence set forth in SEQ ID NO: 90 and a light chain variable sequence set forth in SEQ ID NO: 89, or j. A heavy chain variable sequence set forth in SEQ ID NO: 100, and a light chain variable sequence set forth in SEQ ID NO: 99, or k. The heavy chain variable sequence set forth in SEQ ID NO: 110 and the light chain variable sequence set forth in SEQ ID NO: 109, or l. The above complex comprising the variable sequence of the heavy chain shown in SEQ ID NO: 120 and the variable sequence of the light chain shown in SEQ ID NO: 119.

  Another embodiment of the present invention is a complex as defined above, wherein said antibody is an IgG antibody.

Another embodiment of the present invention is to bind one compound of the following formula, preferably in the form of trifluoroacetate, to a binder peptide or protein residue using transglutaminase , preferably A method for producing a complex as defined above, used in a 2 to 100-fold molar excess with respect to the binder peptide or protein.

[Wherein X ₁ , X ₂ , X ₃ , SG, L 1, R 1 have the same meaning as described above, for example, in formula (IIa), and R ^y represents —H, —C (═O) —NH - alkyl, -NH-C (= O) - _{alkyl, -C (= O) -NH 2} , or -NH _2. ]

[Wherein X ₁ , X ₂ , X ₃ , SG, L 1, R 1 and Rx have the same meaning as defined above. ]
Another embodiment of the present invention is a complex as defined above, wherein said binder peptide or protein represents an antibody or a derivative of a binder peptide or protein according to the formula:

  Another embodiment of the present invention is a pharmaceutical composition comprising a complex as defined above or a compound as defined above in combination with an adjuvant suitable as an inert, non-toxic medicament.

  Another embodiment of the present invention is a complex as defined above or a compound as defined above for use in a method for the treatment and / or prevention of a disease.

  Another embodiment of the invention is a complex as defined above or a compound as defined above for use in a method of treating hyperproliferative and / or angiogenic injury.

  Another embodiment of the present invention is the treatment and / or prevention of hyperproliferative and / or angiogenic injury in humans and animals using an effective amount of at least one complex as defined above or a compound as defined above. Is the method.

A particularly preferred embodiment is a complex according to one of the following formulas: Ak3a, Ak3b, AD3d, Ak3e represents a binder, preferably an antibody, n is 2 to 10, preferably 2 to 4, and further Preferably 2 or 4 is represented.

Therapeutic uses Hyperproliferative diseases in which the compounds according to the invention can be used for treatment include in particular the group of cancer and tumor diseases. In the context of the present invention, these are in particular the following diseases (but not limited to): breast cancer and breast tumors (breast cancers such as ductal and lobular types, also in situ), respiratory tract Tumors (small and non-small cell lung cancer, bronchial cancer), brain tumors (eg brain stem and hypothalamic tumors, astrocytoma, ependymoma, glioblastoma, glioma, medulloblastoma, medulla Meningiomas and neuro-ectodermal and pineal tumors), gastrointestinal tumors (esophageal, stomach, gallbladder, small intestine, large intestine, rectal and anal cancer), liver tumors (especially hepatocellular carcinoma) , Cholangiocarcinoma and mixed hepatocellular cholangiocarcinoma), head and neck tumors (larynx, hypopharynx, nasopharynx, oropharyngeal cancer, lip and oral cancer, oral melanoma), skin tumors (basal cell tumor, spines) Cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, Melanoma, non-melanoma skin cancer, Merkel cell skin cancer, mast cell tumor), soft tissue tumor (especially soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, chondrosarcoma, fibrosarcoma, angiosarcoma, leiomyosarcoma) , Liposarcoma, lymphosarcoma and rhabdomyosarcoma), ocular tumors (especially intraocular melanoma and retinoblastoma), endocrine and exocrine gland tumors (eg thyroid and parathyroid, pancreatic and salivary gland cancers, glands) Cancer), urinary tract tumors (bladder, penis, kidney, renal pelvis and ureteral tumors) and genital tumors (endometrial, cervical, ovarian, vagina, vulva and uterine cancers in women, and in men) Prostate and testicular cancer). These also include proliferative diseases of blood, lymphatic system and spinal cord as solid or circulating cells such as leukemia, lymphoma and myeloproliferative diseases such as acute myeloid, acute lymphoblastic, chronic lymphatic, Chronic myeloid and hairy cell leukemias, and AIDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma and lymphoma in the central nervous system.

  These well-characterized diseases in humans can occur in other mammals with similar etiology and are equally treatable with the compounds of the present invention.

  Treatment of the cancer diseases mentioned above with the compounds according to the invention includes both the treatment of solid tumors and their metastatic and circulating treatments.

  In the context of the present invention, the terms “treatment” or “treating” are used in the conventional sense to fight, reduce, attenuate or ameliorate a disease or health disorder, and for example in the case of cancer As mentioned above, it means that the patient is cared for, cared for, and nursed for the purpose of improving the living conditions that are impaired by this disease.

  The invention therefore further provides the use of the compounds according to the invention for the treatment and / or prevention of disorders, in particular the disorders mentioned above.

  The invention further provides the use of the compounds according to the invention for the manufacture of a medicament for the treatment and / or prevention of disorders, in particular the disorders mentioned above.

  The invention further provides the use of the compounds according to the invention in a method for the treatment and / or prophylaxis of disorders, in particular the disorders mentioned above.

  The present invention further provides a method for treating and / or preventing disorders, in particular the disorders mentioned above, using an effective amount of at least one compound according to the invention.

  The compounds according to the invention can be used alone or optionally in combination with one or more other pharmacologically active substances, provided that the combination does not cause undesirable and unacceptable side effects. The invention therefore further provides a medicament comprising at least one compound according to the invention and one or more further active compounds, in particular for the treatment and / or prevention of the abovementioned disorders.

  For example, the compounds of the present invention can be combined with known anti-hyperproliferative, cytostatic or cytotoxic agents for the treatment of cancer diseases. Examples of suitable combination active compounds include:

  131I-chTNT, abarelix, abiraterone, aclarubicin, adu-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alemtuzumab, alendronate, alitretinoin, altretamine, amifostine, aminoglutethimide, hexylaminolevulinate Amrubicin, Amsacrine, Anastrozole, Ancestim, Anetholedithiolethione, Angiotensin II, Antithrombin III, Aprepitant, Arcitsumab, Algrabine, Arsenic trioxide, Asparaginase, Axitinib, Azacitidine, Basiliximab, Belotecan benstatuto benstatut Bicalutamide, bisantrene, bleomycin, bortezomib, Relin, Bostinib, Brentuximab vedotin, Busulfan, Cabazitaxel, Cabozantinib, Calcium folinate, Calcium levofolinate, capecitabine, capromab, carboplatin, carfilzomib, carmofur, carmustine, clomaximab, celecoxib, celecoxib, celecoxib , Cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, copanricib, chrysantaspase, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, decitabine, decitabine, decitabine, decitaxin Leukin diftitox Denosumab, depreotide, deslorelin, dexrazoloxane, dibrospididium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxyfluridine, doxorubicin, doxorubicin + estrone, dronabinol, eculizumab, edrecolomab Enocitabine, enzalutamide, epirubicin, epithiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, etoposide, everolimus, exemestane, fadrozole, fentanyl, fentanyl, filtanzil Fluoxymesterone, Phlox Uridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetate, gallium nitrate, ganirelix, gefitizumab, gemcitabib (Glutoxim), GM-CSF, goserelin, granisetron, granulocyte colony-stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seed, lansoprazole, ibandronic acid, ibritumomab tiuxetane, ibrutinib, idarubicin, ifosfamide, imatinib , Improsulfan, indisetron, incadronic acid Ingenol mebutate, interferon alpha, interferon beta, interferon gamma, iobitridol, iobenguran (123I), iomeprol, ipilimumab, irinotecan, itraconazole, ixabepilone, lanreotide, lapatinib, lasocholine, lenalidomide, lengradomizole, lennan Prorelin, levamisole, levonogestrel, levothyroxine sodium, lisuride, lovatatin, lomustine, lonidamine, masoprocol, medroxyprogesterone, megestrol, meralsoprole, melphalan, mepithiostan, mercaptopurine, mesna, methadone, methotrexate, methoxalene, Methyl aminolevulinate, methylprednisolone, Tiltestosterone, metyrosine, mifamultide, miltefocin, miriplatin, mitobronitol, mitoguazone, mitractol, mitomycin, mitotane, mitoxantrone, mogamulizumab, morglamostim, mopidamol, morphine hydrochloride, morphine sulfate, nabilone, nabinoximols naroquinolcine , Narutograstim, nedaplatin, nelarabine, neridronate, nivolumabpentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nitracrine, nivolumab, octetuzumab, octetuzumab , Oprelbequin, orgothein, orillothymod, oxaliplatin, oxycodone, oxymetholone, ozogamicin, p53 gene therapy, paclitaxel, palyfermine, palladium-103 seed, palonosetron, pamidronate, panitumumab, pantoprazole, pazopanib, pegaspargine, PEG-epoetin Beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentazocine, pentostatin, pepromycin, perflubutane, perphosphamide, pertuzumab, pisibanil, pilocarpine, pirarubicin, pixantrone, prilixafor, promycin Polyglutam, polyestradiol phosphate, poly Nylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, predonimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, lacotumomab, radium-223, radoxide , Ramosetron, ramucirumab, ranimustine, rasburicase, razoxan, refametinib, regorafenib, risedronate, etidronate rhenium-186, rituximab, romidepsin, romiplostim, romultide, roniccrib, samarium (153Sm) Schizophyllan, sobuzoxane Glycididazole sodium, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, tamibarotene, tamoxifen, tapentadol, tasonermine, teseleukin, technetium (99mTc) nofetumomab merpentane, 99mTc Tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, timalfacin, thioguanine, tocilizumab, topotecan, toremifene, tositumomaz Trifluridine + tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, varatinib, valrubicin, vandetanib, bapreotide, vemurafenib, vinblastine, vincristine, vindezine, vinflunobine, vinflurinbine Spheres, dinostatin, dinostatin stimamarer, zoledronic acid, zorubicin.

  Furthermore, the compounds of the present invention can be combined with binders that can bind to, for example, the following targets: OX-40, CD137 / 4- 1BB, DR3, IDO1 / IDO2, LAG-3, CD40, for example.

  Furthermore, the compounds according to the invention can also be used in combination with radiation therapy and / or surgical intervention.

  In general, the following objectives can be pursued in combination with the compounds of the present invention and other cytostatic or cytotoxic active agents.

An improvement in efficacy in delaying tumor growth, reducing its size, or even in its complete removal compared to treatment with individual active compounds;
The possibility of using chemotherapeutic agents used at lower doses than with monotherapy;
● Possibility of a more tolerated treatment with fewer side effects compared to individual administration;
● Possibility of treatment for tumor diseases with a broader spectrum;
● Achieve higher response speed to therapy;
● Long patient survival compared to modern standard therapies.

  Furthermore, the compounds according to the invention can also be used in combination with radiation therapy and / or surgical intervention.

  The present invention further provides a medicament comprising at least one compound according to the invention together with one or more inert and non-toxic medicaments which are typically suitable as medicaments, and the use thereof for the above purposes.

  The compounds according to the invention can act systemically and / or locally. In this regard, it can be administered in any suitable form, eg parenterally, by inhalation where possible, or as an implant or stent.

  The compounds according to the invention can be administered in administration forms suitable for these administration routes.

  Parenteral administration can avoid absorption steps (eg, intravenous, arterial, intracardiac, intrathecal or lumbar) or absorption (eg, intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal) ) Can be included. Suitable dosage forms for parenteral administration include formulations for injection and infusion in the form of solutions, suspensions, emulsions or lyophilizates. Preference is given to parenteral administration, in particular intravenous administration.

  In general, for parenteral administration, it is advantageous to administer an amount of about 0.001 to 1 mg / kg, preferably about 0.01 to 0.5 mg / kg, to obtain effective results. It has been.

  Even so, it may be necessary to deviate from the specified amount, as appropriate, depending on the specific body weight, route of administration, individual response to the active compound, type of formulation, and time or interval of administration. possible. Thus, in some cases, an amount less than the minimum amount may be sufficient, but in other cases the upper limit mentioned must be exceeded. For larger doses it may be advisable to divide them into several individual doses over the day.

Examples The following examples illustrate the invention. The present invention is not limited to the examples.

  Unless otherwise noted, the percentages in the tests and examples below are percentages by weight and parts are parts by weight. The solvent ratio, dilution ratio and concentration data in the liquid / liquid solution are in each case volume-based.

Synthesis route:
As an example of a working example, the following diagram shows an exemplary synthetic route to a working example.

Scheme 1: Synthesis of glutamine-linked ADC

  n is 2 or 4.

Scheme 2

  a. HATU, DIPEA, DMF, rt; b. ACN, buffer pH 8, room temperature; c. HCl 4M, dioxane, room temperature.

Scheme 3: Synthesis of glutamine-linked ADC

  n is 2 or 4.

Scheme 4

[A): eg benzyl bromide, Cs ₂ CO ₃ , DMF, RT; b) eg Pd (dppf) ₂ Cl ₂ , DMF, Na ₂ CO ₃ , 85 ° C .; c) eg LiAlH ₄ , THF, 0 ° C .; MnO ₂ , DCM, RT; d) eg Ti (iOPr) ₄ , THF, RT; e) eg tBuLi, THF, −78 ° C .; MeOH, NH ₄ Cl; f) eg HCl / 1,4-dioxane]
Scheme 5

a. HATU, DIPEA, DMF, rt; b) Pd / C 10%, MeOH; c) HATU, DIPEA, DMF, rt, d) Pd / C 10%, MeOH
Scheme 6

[A): eg sodium triacetoxyborohydride, acetic acid, DCM, RT; b) eg acetoxyacetyl chloride, NEt ₃ , DCM, RT; c) eg LiOH, THF / water, RT; d) eg H ₂ , Pd -C, EtOH, RT; e) e.g. Teoc-OSu, NEt _3, dioxane, RT; f) e.g. Fmoc-Cl, diisopropylethylamine, dioxane / water 2: 1, RT]
Scheme 7:

[A) HATU, DMF, diisopropylethylamine, RT; b) TFA, DCM, RT; c) HATU, DMF, diisopropylethylamine, RT; d) zinc chloride, trifluoroethanol, 50 ° C .; e) H ₂ , Pd / C, THF / water, RT]
Scheme 8

[A) HATU, DMF, diisopropylethylamine, RT; b) H ₂ , Pd / C, THF / water, RT; c) zinc chloride, trifluoroethanol, 50 ° C.]
Scheme 9: Synthesis of glutamine-linked ADC

  n is 2 or 4.

Scheme 10:

[A): sodium triacetoxyborohydride, acetic acid, DCM, RT; b) acetoxyacetyl chloride, triethylamine, DCM, RT; c) L-cysteine, NaHCO ₃ , DBU, isopropanol / water, RT; d) Boc- 6-aminocaproic acid, HATU, DMF, diisopropylethylamine, RT; e) zinc chloride, trifluoroethanol, 50 ° C., EDTA. ]
A. Example
Abbreviations and acronyms:
A431NS: human tumor cell line A549: human tumor cell line ABCB1: ATP binding cassette subfamily B member 1 (synonymous with P-gp and MDR1)
abs. : Pure Ac: Acetyl ACN: Acetonitrile aq. : Aqueous, aqueous solution ATP: Adenosine triphosphate BCRP: Breast cancer resistance protein, efflux transporter BEP: 2-bromo-1-ethylpyridinium tetrafluoroborate Boc: tert-butoxycarbonyl br. : Wide (by NMR)
Ex. : Example CI: Chemical ionization (by MS)
D: Double line (in NMR)
D: JP TLC: Thin layer chromatography DCI: Direct chemical ionization (by MS)
Dd: Double line double line (in NMR)
DMAP: 4-N, N-dimethylaminopyridine DME: 1,2-dimethoxyethane DMEM: Dulbecco's modified Eagle medium (standard culture for cell culture)
DMF: N, N-dimethylformamide DMSO: Dimethyl sulfoxide DPBS, D-PBS, PBS: Dulbecco's phosphate buffered saline PBS = DPBS = D-PBS, pH 7.4, from Sigma, No D8537
composition:
KCl 0.2g
KH ₂ PO ₄ (anhydride) 0.2 g
NaCl 8.0g
Na ₂ HPO ₄ (anhydride) 1.15 g
1 liter with H ₂ O Dt: triplet doublet (by NMR)
DTT: DL-dithiothreitol EDC: N ′-(3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride EGFR: epidermal growth factor receptor EI: electron impact ionization (in MS)
ELISA: Enzyme-linked immunosorbent assay eq. : Equivalent ESI: Electrospray ionization (in MS)
ESI-MicroTofq: ESI- MicroTofq (Tof = time of flight and q = name of mass spectrometer using quadrupole)
FCS: fetal bovine serum Fmoc: (9H-fluoren-9-ylmethoxy) carbonyl GTP: guanosine 5 'triphosphate H: time HATU: O- (7-azabenzotriazol-1-yl) -N, N, N' , N'-tetramethyluronium hexafluorophosphate HCT-116: human tumor cell line HEPES: 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid HOAc: acetic acid HOAt: 1-hydroxy-7-azabenzotriazole HOBt: 1-hydroxy -1H- benzotriazole hydrate HOSu: N-hydroxysuccinimide HPLC: high pressure high performance liquid chromatography HT29: human tumor cell lines IC _50: half-maximal inhibitory concentration i. m. : Musclely, intramuscular administration i. v. : Intravenously, intravenously conc. : Concentrated LC-MS: Liquid chromatography coupled mass spectrometry LLC-PK1 cells: Lewis lung cancer pork kidney cell line L-MDR: Human MDR1 transfected LLC-PK1 cells M: Multiplex (by NMR)
MDR1: Multidrug resistance protein 1
MeCN: acetonitrile Me: methyl Min: min MS: mass spectrometry MTT: 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide 3
NCI-H292: human tumor cell line NCI-H520: human tumor cell line NMM: N-methylmorpholine NMP: N-methyl-2-pyrrolidinone NMR: nuclear magnetic resonance spectrum measurement NMRI: mouse derived from Naval Medical Research Institute (NMRI) Strain NSCLC: Non-small cell lung cancer PBS: Phosphate buffer solution Pd / C: Palladium / activated charcoal P-pg: P-glycoprotein, transporter protein PNGaseF: enzyme that cleaves sugar quant. : Quantitative (in yield)
Quart: Quadruple (by NMR)
Quint: Quintet (by NMR)
R _f : Retention index (in TLC)
RT: room temperature R _t : retention time (by HPLC)
S: Single line (in NMR)
s. c. : Subcutaneous, subcutaneous administration SCC-4: human tumor cell line SCC-9: human tumor cell line SCID mice: test mice with severe combined immunodeficiency t: triplet (by NMR)
TBAF: tetra-n-butylammonium fluoride TEMPO: (2,2,6,6-tetramethylpiperidin-1-yl) oxyl tert: tertiary TFA: trifluoroacetic acid THF: tetrahydrofuran T3P (registered trademark): 2, 4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinan 2,4,6-trioxide UV: UV spectrum measurement v / v: volume ratio (in solution)
Z: benzyloxycarbonyl.

Amino acid abbreviations Ala = alanine Arg = arginine Asn = asparagine Asp = aspartic acid Cys = cysteine Glu = glutamic acid Gln = glutamine Gly = glycine His = histidine Ile = isoleucine Leu = leucine Lys = lysine Met = methionine Nv = methionine Proline Ser = serine Thr = threonine Trp = tryptophan Tyr = tyrosine Val = valine.

  In the context of this disclosure, room temperature should always be assumed if no temperature is given in the description of the reaction.

HPLC method and LC-MS method:
Method 1 (LC-MS) :
Apparatus: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8μ 50 × 1 mm; Gradient: 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A Oven: 50 ° C .; flow rate: 0.40 mL / min; UV detection: 208-400 nm.

Method 2 (LC-MS):
MS apparatus type: Waters Synapt G2S; UPLC apparatus type: Waters Acquity I-CLASS; column: Waters, BEH300, 2.1 × 150 mm, C18 1.7 μm; mobile phase A: 1 liter of water + 0.01% formic acid; mobile phase B: 1 liter of acetonitrile + 0.01% formic acid; Gradient: 0.0 min 2% B → 1.5 min 2% B → 8.5 min 95% B → 10.0 min 95% B; Oven: 50 ° C .; Flow rate: 0.50 mL / min; UV detection: 220 nm.

Method 3 (LC-MS):
MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100 series; Column: Agilent ZORBAX Extended-C18 3.0 x 50 mm 3.5 micron; Mobile phase A: 1 liter of water + 0.01 mol ammonium carbonate, mobile phase B : 1 liter of acetonitrile; gradient: 0.0 min 98% A → 0.2 min 98% A → 3.0 min 5% A → 4.5 min 5% A; oven: 40 ° C .; flow rate: 1.75 mL / Minute; UV detection: 210 nm.

Method 4 (LC-MS) :
MS apparatus type: Waters Synapt G2S; UPLC apparatus type: Waters Acquity I-CLASS; column: Waters, HSST3, 2.1 × 50 mm, C18 1.8 μm; mobile phase A: 1 liter of water + 0.01% formic acid; mobile phase B: 1 liter of acetonitrile + 0.01% formic acid; Gradient: 0.0 min 10% B → 0.3 min 10% B → 1.7 min 95% B → 2.5 min 95% B; Oven: 50 ° C .; Flow rate: 1.20 mL / min; UV detection: 210 nm.

Method 5 (LC-MS):
Apparatus: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8μ 50 × 1 mm; Gradient: 0.0 min 95% A → 6.0 min 5% A → 7.5 min 5% A Oven: 50 ° C .; Flow rate: 0.35 mL / min; UV detection: 210-400 nm.

Method 6 (LC-MS) :
Apparatus: Micromass Quattro Premier equipped with Waters UPLC Acquity; Column: Thermo Hypersil GOLD 1.9 μ50 × 1 mm; Mobile phase A: 1 liter of water + 0.5% of 50% strength formic acid, Mobile phase B: 1 liter of acetonitrile + 0.5 mL of 50% strength formic acid; Gradient: 0.0 min 97% A → 0.5 min 97% A → 3.2 min 5% A → 4.0 min 5% A Oven: 50 ° C .; flow rate: 0.3 mL / min; UV detection: 210 nm .

Method 7 (LC-MS):
Equipment: Agilent MS Quad 6150; HPLC: Agilent 1290; Column: Waters Acquity UPLC HSS T3 1.8μ 50 × 2.1 mm; Mobile Phase A: 1 liter of water + 0.25 mL of 99% strength formic acid, Mobile Phase B: 1 liter of acetonitrile + 0.25% strength formic acid 0.25 mL; Gradient: 0.0 min 90% A → 0.3 min 90% A → 1.7 min 5% A → 3.0 min 5% A Oven: 50 ° C .; 20 mL / min; UV detection: 205-305 nm.

Method 8 (LC-MS) :
MS apparatus type: Waters Synapt G2S; UPLC apparatus type: Waters Acquity I-CLASS; column: Waters, HSST3, 2.1 × 50 mm, C18 1.8 μm; mobile phase A: 1 liter of water + 0.01% formic acid; mobile phase B: 1 liter of acetonitrile + 0.01% formic acid; gradient: 0.0 min 2% B → 2.0 min 2% B → 13.0 min 90% B → 15.0 min 90% B; oven: 50 ° C .; Flow rate: 1.20 mL / min; UV detection: 210 nm.

Method 9 : LC-MS-Prep purification method for Examples 181 to 191 (Method LIND-LC-MS-Prep)
MS apparatus: Waters, HPLC apparatus: Waters (column Waters X-Bridge C18, 19 mm × 50 mm, 5 μm, mobile phase A: water + 0.05% ammonia, mobile phase B: acetonitrile (ULC) gradient; flow rate: 40 mL / min; UV detection: DAD; 210-400 nm).

Or MS apparatus: Waters, HPLC apparatus: Waters (column Phenomenex Luna 5μ C18 (2) 100A, AXIA Tech. 50 × 21.2 mm, mobile phase A: water + 0.05% formic acid, mobile phase B: acetonitrile (ULC) gradient Flow rate: 40 mL / min; UV detection: DAD; 210-400 nm).

Method 10 : LC-MS analysis method for Examples 181 to 191 (LIND_SQD_SB_AQ)
MS instrument: Waters SQD; HPLC instrument: Waters UPLC; column: Zortex SB-Aq (Agilent), 50 mm x 2.1 mm, 1.8 μm; mobile phase A: water + 0.025% formic acid, mobile phase B: acetonitrile (ULC ) + 0.025% formic acid; gradient: 0.0 min 98% A-0.9 min 25% A-1.0 min 5% A-1.4 min 5% A-1.41 min 98% A-1 Oven: 40 ° C .; Flow rate: 0.600 mL / min; UV detection: DAD; 210 nm.

Method 11 (HPLC):
Apparatus: HP1100 series Column: Merck Chromolith SpeedROD RP-18e, 50-4.6 mm, catalog number 1.51450.0001, Precolumn Chromolith Guard cartridge kit, RP-18e, 5-4.6 mm, catalog number 1.51470.0001
Gradient: flow rate 5 mL / min
Injection volume 5μL
Solvent A: HClO ₄ (70% strength) / water (4 mL / L)
Solvent B: acetonitrile 20% B starting
0.50min 20% B
3.00 minutes 90% B
3. 50% 90% B
3.51 min 20% B
4.00min 20% B
Column temperature: 40 ° C
Wavelength: 210 nm.

  All reactants or reagents whose manufacture is not explicitly described below are commercially purchased from commonly available sources. For all other reactants or reagents that are also not described below and are not commercially available or obtained from sources that are not normally available, the publications describing their manufacture are also described. Are listed.

Method 12 (LC-MS)
Instrument MS: Thermo Scientific FT-MS; Instrument UHPLC +: Thermo Scientific UltimateMate 3000; Column: Waters, HSST3, 2.1 × 75 mm, C18 1.8 μm; Eluent A: 1 liter of water + 0.01% formic acid; Eluent B : Acetonitrile 1 liter + 0.01% formic acid; gradient: 0.0 min 10% B → 2.5 min 95% B → 3.5 min 95% B; oven: 50 ° C .; flow rate: 0.90 mL / min; UV Detection: 210 nm / optimal integration path 210-300 nm.

Method 13 : (LC-MS)
Equipment MS: Waters (Micromass) Quattro Micro; Equipment Waters UPLC Acquity; Column: Waters BEHC18 1.7 μ 50 × 2.1 mm; Eluent A: 1 liter of water + 0.01 mol ammonium formate, Eluent B: 1 liter of acetonitrile; Gradient : 0.0 minutes 95% A → 0.1 minutes 95% A → 2.0 minutes 15% A → 2.5 minutes 15% A → 2.51 minutes 10% A → 3.0 minutes 10% A; oven : 40 ° C; flow rate: 0.5 mL / min; UV detection: 210 nm.

Method 14: (LC-MS):
Raw materials and intermediates:
Intermediate C1
Trifluoroacetic acid- (1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropan-1-amine (1: 1)

  The title compound was prepared according to the method described in WO2006 / 002326.

Intermediate C2
tert-butyl- (2S) -4-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} Amino) -2-[(tert-butoxycarbonyl) amino] butanoate

Dissolve 4.22 g (14.5 mmol) of tert-butyl N- (tert-butoxycarbonyl) -L-homoselinate in 180 mL of dichloromethane, and add 3.5 mL of pyridine and 1,1,1-triacetoxy-1λ ⁵ , 2-benzoiodine. 9.2 g (21.7 mmol) of xol-3 (1H) -one was added. The reaction is stirred at room temperature for 1 hour, then diluted with 500 mL of dichloromethane, extracted twice with 10% strength sodium thiosulfate solution, twice with 5% strength citric acid and twice with 10% strength sodium bicarbonate solution. Extracted in that order. The organic phase was separated, dried over magnesium sulfate and dried in vacuo. The residue was taken up in diethyl ether and HCl (solution in diethyl ether) was added. The precipitate was filtered off and the filtrate was concentrated and lyophilized from acetonitrile / water. This gave 3.7 g (93%) of tert-butyl (2S) -2-[(tert-butoxycarbonyl) amino] -4-oxobutanoate which was used in the next step without further purification. ( _Rf : 0.5 (DCM / methanol 95/5)).

  Intermediate C1 3.5 g (9.85 mmol) was dissolved in DCM 160 mL and sodium triacetoxyborohydride 3.13 g (14.77 mmol) and acetic acid 0.7 mL were added. After stirring at room temperature for 5 minutes, 3.23 g (11.85 mmol) of tert-butyl (2S) -2-[(tert-butoxycarbonyl) amino] -4-oxobutanoate was added, and the reaction solution was further stirred at room temperature for 30 minutes. Stir for minutes. The solvent was distilled off under reduced pressure and the residue was taken up in acetonitrile / water. The precipitated solid was filtered and dried to give 5.46 g (84%) of the title compound.

HPLC (Method 11): R _t = 2.5 min;
LC-MS (method _1): R t = 1.13 min; MS (ESIpos): m / z = 613 (M + H) +.

Intermediate C3
(2S) -4-[(acetoxyacetyl) {(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} Amino] -2-[(tert-butoxycarbonyl) amino] butanoic acid

  Intermediate C2 (5.46 g, 8.24 mmol) was dissolved in DCM (160 mL), and triethylamine (4.8 mL) and acetoxyacetyl chloride (2.2 mL, 20.6 mmol) were added. The reaction was stirred at room temperature overnight and concentrated under reduced pressure. The residue was taken up in ethyl acetate and extracted three times with saturated sodium bicarbonate solution and then with saturated sodium chloride solution. The organic phase was dried over sodium sulfate and concentrated. The residue was purified by column chromatography on Biotage / Isolera (SNAP 340 g) using the mobile phase cyclohexane / ethyl acetate 2: 1. This gave 4.57 g (75%) of the acylated intermediate.

LC-MS (method _1): R t = 1.49 min; MS (ESIpos): m / z = 713 (M + H) +.

1 g (1.36 mmol) of this intermediate was dissolved in 20 mL of DCM and 20 mL of TFA was added. After stirring at room temperature for 5 hours, the mixture was concentrated and the residue was triturated twice with n-pentane. In each case, n-pentane was removed by decantation and the remaining solid was dried under high vacuum. This gives (2S) -4-[(acetoxyacetyl) {(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2- 1.1 g of dimethylpropyl} amino] -2-aminobutanoic acid / trifluoroacetic acid (1: 1) was obtained. LC-MS (method _1): R t = 0.93 min; MS (ESIpos): m / z = 557 (M + H) +.

  0.91 g (1.57 mmol) of this intermediate was dissolved in 70 mL of DCM, and 3.43 g (15.7 mmol) of di-tert-butyl dicarbonate and 4.1 mL of N, N-diisopropylethylamine were added. After stirring at room temperature for 30 minutes, the reaction was diluted with DCM and extracted with 5% strength citric acid. The organic phase was dried over sodium sulfate and concentrated. The residue was triturated twice with n-pentane, and in each case n-pentane was removed by decantation. The remaining solid was lyophilized from acetonitrile / water 1: 1 to give 1.11 g of the title compound.

HPLC (Method 11): R _t = 2.55 min;
LC-MS (Method 1): R _t = 1.3 min; MS (ESIpos): m / z = 657 (M + H) ⁺ .

Intermediate C4
(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} (Glycoloyl) amino] butanoic acid / trifluoroacetic acid (1: 1)

  Intermediate C2 (5.46 g, 8.24 mmol) was dissolved in DCM (160 mL), and triethylamine (4.8 mL) and acetoxyacetyl chloride (2.2 mL, 20.6 mmol) were added. The reaction was stirred at room temperature overnight and concentrated under reduced pressure. The residue was taken up in ethyl acetate and extracted three times with saturated sodium bicarbonate solution and then with saturated sodium chloride solution. The organic phase was dried over sodium sulfate and concentrated. The residue was purified by column chromatography on Biotage / Isolera (SNAP 340 g) using the mobile phase cyclohexane / ethyl acetate 2: 1. This gave 4.57 g (75%) of the acylated intermediate.

LC-MS (method _1): R t = 1.49 min; MS (ESIpos): m / z = 713 (M + H) +.

  1.5 g (2.035 mmol) of this intermediate was taken up in 50 mL of ethanol and 5.8 mL of 40% strength aqueous methanamine solution was added. The reaction was stirred at 50 ° C. for 4 hours and concentrated. The residue was taken up in DCM and washed twice with water. The organic phase was dried over magnesium sulfate and concentrated.

  The residue was high vacuum dried. This gave 1.235 mg of this intermediate, which was reacted further without further purification.

  1.235 mg (1.5 mmol) of this intermediate was dissolved in 15 mL DCM and 15 mL TFA was added. After stirring at room temperature for 4 hours, the mixture was concentrated. The residue was purified by preparative HPLC. Appropriate fractions were concentrated and the residue was lyophilized from acetonitrile. This gave 1.04 g (quantitative) of the title compound.

HPLC (Method 11): R _t = 1.9 min;
LC-MS (method _1): R t = 0.89 min; MS (ESIpos): m / z = 515 (M + H) +.

Intermediate C5
(2S) -4-[{(1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino ] -2-[(tert-butoxycarbonyl) amino] butanoic acid

  Intermediate C4 (0.9 g, 1.24 mmol) was dissolved in DCM (60 mL), and di-tert-butyl dicarbonate (2.7 g, 12.5 mmol) and N, N-diisopropylethylamine (3.3 mL) were added. After stirring for 45 minutes at room temperature, the reaction was concentrated, the residue was taken up in diethyl ether and n-pentane was added until the mixture began to become cloudy. The reaction solution was cooled to 0 ° C., and the solution was removed by a gradient method. Again, n-pentane was added to the residue and the mixture was decanted to remove liquid. The remaining solid was lyophilized from acetonitrile / water 1: 1 to give 0.95 g (quantitative) of the title compound.

HPLC (Method 11): R _t = 2.5 min;
LC-MS (method _1): R t = 1.27 min; MS (ESIpos): m / z = 615 (M + H) +.

Intermediate C52
(1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropan-1-amine

  First, 10.00 g (49.01 mmol) of methyl 4-bromo-1H-pyrrole-2-carboxylate was placed in 100.0 mL of DMF, and 20.76 g (63.72 mmol) of cesium carbonate and 9.22 g of benzyl bromide (53 .91 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned between water and ethyl acetate and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over magnesium sulfate and the solvent was removed under reduced pressure. The reaction was repeated with 90.0 g of methyl 4-bromo-1H-pyrrole-2-carboxylate.

  The two combined reaction acquisitions were purified by preparative RP-HPLC (column: Daiso 300 × 100; 10μ, flow rate: 250 mL / min, MeCN / water). The solvent was removed under reduced pressure and the residue was dried under high vacuum. This gave 125.15 g (87% of theory) of the compound 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate.

LC-MS (method _1): R t = 1.18 min; MS (ESIpos): m / z = 295 [M + H] +.

  First, under argon, 4.80 g (16.32 mmol) of methyl 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate was placed in DMF and 3.61 g of (2,5-difluorophenyl) boronic acid. (22.85 mmol), saturated sodium carbonate 19.20 mL solution and [1,1′-bis (diphenylphosphino) ferrocene] -dichloropalladium (II): dichloromethane 1.33 g (1.63 mmol) were added. The reaction mixture was stirred at 85 ° C. overnight. The reaction mixture was filtered through celite and the filter cake was washed with ethyl acetate. The organic phase was extracted with water and washed with saturated NaCl solution. The organic phase was dehydrated with magnesium sulfate and the solvent was distilled off under reduced pressure. The residue was purified on silica gel (mobile phase: cyclohexane / ethyl acetate 100: 3). The solvent was removed under reduced pressure and the residue was dried under high vacuum. This gave 3.60 g (67% of theory) of the compound 1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole-2-carboxylate.

LC-MS (method _7): R t = 1.59 min; MS (ESIpos): m / z = 328 [M + H] +.

  First, 3.60 g (11.00 mmol) of methyl 1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole-2-carboxylate was placed in 90.0 mL of THF, and 1.04 g of lithium aluminum hydride was added. (27.50 mmol) (2.4 M solution in THF) was added at 0 ° C. The reaction mixture was stirred at 0 ° C. for 30 minutes. At 0 ° C., saturated potassium sodium tartrate solution was added and ethyl acetate was added to the reaction mixture. The organic phase was extracted 3 times with saturated sodium potassium tartrate solution. The organic phase was washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was dissolved in 30.0 mL of dichloromethane. Manganese (IV) oxide 3.38 g (32.99 mmol) was added and the mixture was stirred at room temperature for 48 hours. Additional 2.20 g (21.47 mmol) manganese (IV) oxide was added and the mixture was stirred at room temperature overnight. The reaction mixture was filtered through celite and the filter cake was washed with dichloromethane. The solvent was distilled off under reduced pressure, and 2.80 g of the residue (1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole-2-carbaldehyde) was obtained in the next synthesis without further purification. Used in stages.

LC-MS (method _7): R t = 1.48 min; MS (ESIpos): m / z = 298 [M + H] +.

  First, 28.21 g (94.88 mmol) of 1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole-2-carbaldehyde and 23.00 g of (R) -2-methylpropane-2-sulfinamide (189.77 mmol) was taken up in 403.0 mL of pure THF, 7.42 g (237.21 mmol) of titanium (IV) isopropoxide was added and the mixture was stirred at room temperature overnight. Saturated NaCl solution 500.0 mL and ethyl acetate 1000.0 mL were added and the mixture was stirred at room temperature for 1 hour. The mixture was filtered through diatomaceous earth and the filtrate was washed twice with saturated NaCl solution. The organic phase was dehydrated with magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified using Biotage Isolara (silica gel, column 1500 + 340 g SNAP, flow rate 200 mL / min, ethyl acetate / cyclohexane 1:10).

LC-MS (Method 7): R _t = 1.63 min; MS (ESIpos): m / z = 401 [M + H] ⁺ .

  First (R) -N-{(E / Z)-[1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] methylene} -2-methylpropane-2-sulfine 25.00 g (62.42 mmol) of amide was placed in pure THF under argon and cooled to −78 ° C. 12.00 g (187.27 mmol) of tert-butyllithium (1.7 M solution in pentane) was added at −78 ° C. and the mixture was stirred at this temperature for 3 hours. At −78 ° C., 71.4 mL of methanol and 214.3 mL of saturated ammonium chloride solution were added in that order, and the reaction mixture was warmed to room temperature and stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dehydrated with magnesium sulfate and the solvent was distilled off under reduced pressure. Residue (R) -N-{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} -2- Methylpropane-2-sulfinamide was used in the next synthetic step without further purification.

LC-MS (method _6): R t = 2.97 min; MS (ESIpos): m / z = 459 [M + H] +.

  First, (R) -N-{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} -2- Methylpropane-2-sulfinamide 28.00 g (61.05 mmol) was placed in 186.7 mL of 1,4-dioxane, and 45.8 mL of HCl / 1,4-dioxane solution (4.0 M) was added. The reaction mixture was stirred at room temperature for 2 hours and the solvent was distilled off under reduced pressure. The residue was purified by preparative RP-HPLC (column: Kinetix 100 × 30; flow rate: 60 mL / min, MeCN / water). Acetonitrile was distilled off under reduced pressure and dichloromethane was added to the aqueous residue. The organic phase was washed with sodium bicarbonate solution and dried over magnesium sulfate. The solvent was removed under reduced pressure and the residue was dried under high vacuum. This gave 16.2 g (75% of theory) of the title compound.

LC-MS (Method 6): R _t = 2.10 min; MS (ESIpos): m / z = 338 [M-NH ₂ ] ⁺ , 709 [2M + H] ⁺ .

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.87 (s, 9H), 1.53 (s, 2H), 3.59 (s, 1H), 5.24 (d 2H), 6.56 (s, 1H), 6.94 (m, 1H), 7.10 (d, 2H), 7.20 (m, 1H), 7.26 (m, 2H), 7 .34 (m, 2H), 7.46 (m, 1H).

Intermediate C53
(2S) -4-[{(1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino ] -2-{[(9H-fluoren-9-ylmethoxy) carbonyl] amino} butanoic acid

  First, similar to C2, intermediate C52 was reductively alkylated with benzyl (2S) -2-{[(benzyloxy) carbonyl] amino} -4-oxobutanoate. The secondary amino group was acylated with ethyl 2-chloro-2-oxoacetate and then the two ester groups were hydrolyzed with 2M lithium hydroxide solution / methanol. The intermediate thus obtained was dissolved in ethanol, palladium / carbon (10%) was added and the mixture was hydrogenated with hydrogen at room temperature and standard pressure for 1 hour. The deprotected compound was taken up in dioxane / water 2: 1 and in the last stage the Fmoc protecting group was introduced using N, N-diisopropylethylamine in the presence of 9H-fluoren-9-ylmethylchlorocarbonate.

LC-MS (Method 1): R _t = 1.37 min; MS (ESIpos): m / z = 734 (M−H) ⁻ .

Intermediate C58
(2S) -4-[{(1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino ] -2-({[2- (Trimethylsilyl) ethoxy] carbonyl} amino) butanoic acid

  Initially, intermediate C52 was reductively alkylated with benzyl (2S) -2-{[(benzyloxy) carbonyl] amino} -4-oxobutanoate in the same manner as intermediate C2. First, similar to C2, intermediate C52 was reductively alkylated with benzyl (2S) -2-{[(benzyloxy) carbonyl] amino} -4-oxobutanoate. The secondary amino group was acylated with ethyl 2-chloro-2-oxoacetate following the procedure described for intermediate C27 and the two ester groups were hydrolyzed with 2M lithium hydroxide solution / methanol. The intermediate thus obtained was dissolved in ethanol, palladium / carbon (10%) was added, and the mixture was hydrogenated with hydrogen at room temperature under standard pressure for 1 hour.

  500 mg (0.886 mmol) of this fully deprotected intermediate is taken up in 60 mL of dioxane and 253 mg (0.975 mmol) of 1-({[2- (trimethylsilyl) ethoxy] carbonyl} oxy) pyrrolidine-2,5-dione. And 198 μL of triethylamine was added. After stirring at room temperature for 24 hours, the reaction was concentrated and the residue was purified by preparative HPLC. Appropriate fractions were combined, concentrated under reduced pressure, and dried under vacuum to give 312 mg (50% of theory) of the title compound.

LC-MS (Method 5): R _t = 4.61 min; MS (ESIpos): m / z = 658 (M + H) ⁻ .

Intermediate C61
N-[(2S) -4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} ( Glycoroyl) amino] -2-({[2- (trimethylsilyl) ethoxy] carbonyl} amino) butanoyl] -β-alanine

  The title compound was prepared by coupling 60 mg (0.091 mmol) of Intermediate C58 with methyl β-alaninate, followed by ester cleavage with 2M lithium hydroxide solution. This gave 67 mg (61% of theory) of the title compound in two steps.

LC-MS (method _1): R t = 1.29 min; MS (ESIpos): m / z = 729 (M + H) +.

Intermediate C69
11-{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} -2,2-dimethyl-6 12-Dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecan-17-one acid

  First (2- (trimethylsilyl) ethyl {3-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethyl 117.0 mg (0.19 mmol) of propyl} (chloroacetyl) amino] propyl} carbamate (intermediate C70) and 21.6 mg (0.20 mmol) of 3-sulfanylpropanoic acid were added to 3.0 mL of methanol, and potassium carbonate 89 0.5 mg (0.65 mmol) was added and the mixture was stirred for 4 hours at 50 ° C. The reaction mixture was diluted with ethyl acetate, the organic phase was washed with water and saturated NaCl solution, the organic phase was dried over magnesium sulfate, The solvent was distilled off under reduced pressure and the residue was dried under high vacuum, which was used in the next synthetic step without further purification. To give the title compound 106.1mg (73% of theory).

LC-MS (Method 1): R _t = 1.42 min; MS (ESIneg): m / z = 700 (M−H) ⁻ .

Intermediate C70
(2- (Trimethylsilyl) ethyl {3-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (Chloroacetyl) amino] propyl} carbamate

  First, (1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropan-1-amine (intermediate C52) 990. 0 mg (2.79 mmol) was placed in 15.0 mL dichloromethane, 828.8 mg (3.91 mmol) sodium triacetoxyborohydride and 129.9 mg (3.21 mmol) acetic acid were added and the mixture was stirred at room temperature for 5 minutes . 698.1 mg (3.21 mmol) of 2- (trimethylsilyl) ethyl (3-oxopropyl) carbamate (Intermediate L15) dissolved in 15.0 mL of dichloromethane was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and the organic phase was washed in each case twice with saturated sodium carbonate solution and with saturated NaCl solution. The organic phase was dehydrated with magnesium sulfate and the solvent was distilled off under reduced pressure. The residue was purified using silica gel (mobile phase: dichloromethane / methanol = 100: 2). The solvent was removed under reduced pressure and the residue was dried under high vacuum. This gave the compound 2- (trimethylsilyl) ethyl [3-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2- 1.25 g (73% of theory) of dimethylpropyl} amino) propyl] carbamate were obtained.

LC-MS (method _1): R t = 1.09 min; MS (ESIpos): m / z = 556 (M + H) +.

  First 2- (trimethylsilyl) ethyl [3-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl } Amino) propyl] carbamate 908.1 mg (1.63 mmol) and triethylamine 545.6 mg (5.39 mmol) were placed in 10.0 mL dichloromethane and the mixture was cooled to 0 ° C. At this temperature, 590.5 mg (5.23 mmol) of chloroacetyl chloride was added and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and the organic phase was washed 3 times with saturated sodium bicarbonate solution and saturated ammonium chloride solution in each case. The organic phase was washed with saturated NaCl solution and dried over magnesium sulfate. The residue was purified by preparative RP-HPLC (column: Reprosil 250 × 30; 10μ, flow rate: 50 mL / min, MeCN / water, 0.1% TFA). The solvent was removed under reduced pressure and the residue was dried under high vacuum. This gave 673.8 mg (65% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.53 min; MS (ESIneg): m / z = 676 (M + HCOO ⁻ ) ⁻ .

Intermediate C71
S- (11-{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} -2,2-dimethyl -6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl) -L-cysteine / trifluoroacetic acid (1: 1)

  536.6 mg (4.43 mmol) of L-cysteine was suspended in 2.5 mL of water and 531.5 mg (6.33 mmol) of sodium bicarbonate. 2- (Trimethylsilyl) ethyl {3-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2 dissolved in 25.0 mL of isopropanol , 2-dimethylpropyl} (chloroacetyl) amino] propyl} carbamate (intermediate C70) 400.0 mg (0.63 mmol) and 1,8-diazabicyclo [5.4.0] undec-7-ene 1.16 g ( 7.59 mmol) was added. The reaction mixture was stirred at 50 ° C. for 1.5 hours. Ethyl acetate was added to the reaction mixture and the organic phase was washed repeatedly with saturated sodium bicarbonate solution and once with saturated NaCl solution. The organic phase was dehydrated with magnesium sulfate, the solvent was distilled off under reduced pressure and the residue was dried under high vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 250 × 30; 10μ, flow rate: 50 mL / min, MeCN / water, 0.1% TFA). The solvent was removed under reduced pressure and the residue was dried under high vacuum. This gave 449.5 mg (86% of theory) of the title compound.

LC-MS (method _1): R t = 1.20 min; MS (ESIpos): m / z = 717 (M + H) +.

Intermediate C74
2- (Trimethylsilyl) ethyl 3-amino-N-[(2S) -4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl ] -2,2-dimethylpropyl} (glycoloyl) amino] -2-({[2- (trimethylsilyl) ethoxy] carbonyl} amino) butanoyl] -D-alaninate trifluoroacetate (1: 1)

  Intermediate C58 75 mg (0.114 mmol) was dissolved in 12.5 mL DMF and coupled with 78 mg (0.171 mmol) intermediate L6 in the presence of HATU 65 mg (0.11 mmol) and N, N-diisopropylethylamine 79 μL. . After purification by preparative HPLC, the residue was dissolved in 20 mL of ethanol and hydrogenated at room temperature for 1 hour in the presence of 10% Pd / C at room temperature. After filtration of the catalyst, the solvent was distilled off under reduced pressure. The residue was purified by preparative HPLC and lyophilized to give 63 mg (64% over 2 steps) of the title compound.

LC-MS (method _1): R t = 1.16 min; MS (EIpos): m / z = 844 [M + H] +.

Intermediate C75
Methyl (2S) -4-[(acetoxyacetyl) {(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl } Amino] -2-({[2- (trimethylsilyl) ethoxy] carbonyl} amino) butanoate

  To a solution of 4.3 g (12.2 mmol) of intermediate C52 in DCM (525 mL) was added 3.63 g (17.12 mmol) sodium triacetoxyborohydride and 8.4 mL acetic acid. After stirring the mixture at room temperature for 5 minutes, methyl- (2S) -4-oxo-2-({[2- (trimethylsilyl) ethoxy] carbonyl} amino) butanoate (using the classical method, (3S) -3-amino (Prepared from -4-methoxy-4-oxobutanoic acid) A solution of 3.23 g (11.85 mmol) in DCM (175 mL) was added and the reaction mixture was stirred at room temperature for 45 min. The crude mixture was diluted with DCM and washed twice with 100 mL saturated sodium bicarbonate solution and then with brine. The organic layer was dehydrated with magnesium sulfate and evaporated. The residue was purified by preparative HPLC to give methyl (2S) -4-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] There were obtained 4.6 g (61%) of -2,2-dimethylpropyl} amino) -2-({[2- (trimethylsilyl) ethoxy] carbonyl} amino) butanoate.

LC-MS (method _12): R t = 1.97 min; MS (ESIpos): m / z = 614.32 (M + H) +.

  Methyl (2S) -4-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} amino)- To a solution of 200 mg (0.33 mmol) 2-({[2- (trimethylsilyl) ethoxy] carbonyl} amino) butanoate in DCM (10 mL) was added 105 μL triethylamine and 77 μL (0.717 mmol) acetoxyacetyl chloride. The reaction mixture was stirred at room temperature overnight, diluted with ethyl acetate and washed twice with saturated sodium bicarbonate solution and then with brine. The organic layer was dehydrated with magnesium sulfate and evaporated to give 213 mg (75%) of the title compound.

LC-MS (method _1): R t = 1.46 min; MS (ESIpos): m / z = 714 (M + H) +.

Intermediate C88
tert-butyl (3R) -3-[({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} Amino) methyl] pyrrolidine-1-carboxylate trifluoroacetate (1: 1)
(Mixture of stereoisomers)

  tert-butyl (3R) -3-[({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} Amino) methyl] pyrrolidine-1-carboxylate trifluoroacetate (1: 1) (Intermediate C52) in a solution of 2.04 mg (5.75 mmol) in dichloromethane (51 mL) was added 1.71 g sodium triacetoxyborohydride ( 8.05 mmol) and 0.40 g (6.61 mmol) of acetic acid were added and the reaction mixture was stirred at room temperature for 5 minutes. A solution of 1.32 g (6.61 mmol) tert-butyl 3-formylpyrrolidine-1-carboxylate in dichloromethane (20 mL) was added and the mixture was stirred at room temperature overnight. Ethyl acetate was added and the organic phase was washed with saturated sodium carbonate solution and brine. The organic phase was dehydrated with magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was dried under high vacuum. The residue was used in the next synthetic step without further purification. This gave 1.86 g (50% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.99 min; MS (ESIpos): m / z = 538 (M + H—CF ₃ CO ₂ H) ⁺ .

Intermediate C89
tert-butyl (3R) -3-{[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (Chloroacetyl) amino] methyl} pyrrolidine-1-carboxylate

  Tert-Butyl (3R) -3-[({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2 with 4 入 れ molecular sieves , 2-dimethylpropyl} amino) methyl] pyrrolidine-1-carboxylate (Intermediate C88) in a solution of 2.89 g (4.19 mmol, 80% purity) in dichloromethane (42 mL) 1.36 g (13.42 mmol) of triethylamine. ) And 2.13 g (18.87 mmol) of chloroacetyl chloride. The reaction mixture was stirred at room temperature for 5 hours and the solvent was distilled off. The residue was purified by preparative HPLC to give 449 mg (17% of theory) and 442 mg (17% of theory) of isomer 1 of the title compound.

Isomer 1 LC-MS (Method 1): R _t = 2.74 min; MS (ESIpos): m / z = 614 (M + H) ⁺ .

Isomer 2 LC-MS (Method 1): R _t = 2.78 min; MS (ESIpos): m / z = 614 (M + H) ⁺ .

Intermediate C90
S- [2-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} {[(3R) -1- (tert-butoxycarbonyl) pyrrolidin-3-yl] methyl} amino) -2-oxoethyl] -L-cysteine (isomer 1)

  To a solution of 493 mg (4.07 mmol) of L-cysteine in water (2.3 mL) was added 489 mg (5.82 mmol) of sodium bicarbonate, followed by tert-butyl (3R) -3-{[{(1R) -1 -[1-Benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (chloroacetyl) amino] methyl} pyrrolidine-1-carboxylate (intermediate) C89, isomer 1) 357 mg (0.58 mmol) in isopropanol (23.0 mL) and 1,8-diazabicyclo (5.4.0) undec-7-ene 1.06 g (6.98 mmol) were added. . The reaction mixture was stirred at 50 ° C. for 3 hours. Ethyl acetate was added and the organic phase was washed with saturated sodium carbonate solution and brine. The organic phase was dehydrated with magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was dried under high vacuum. The residue was used in the next synthetic step without further purification. This gave 255 mg (62% of theory) of the title compound.

LC-MS (method _1): R t = 1.09 min; MS (ESIpos): m / z = 699 (M + H) +.

Intermediate C95
3-{[2-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} {[(3R ) -1- (tert-Butoxycarbonyl) pyrrolidin-3-yl] methyl} amino) -2-oxoethyl] sulfanyl} propanoic acid (isomer 1)

  tert-butyl (3R) -3-{[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (Chloroacetyl) amino] methyl} pyrrolidine-1-carboxylate (intermediate C89, isomer 1) 384.0 mg (0.62 mmol) and 3-sulfanylpropanoic acid 73.0 mg (0.69 mmol) in methanol (14 mL) And 302.5 mg (2.19 mmol) of potassium carbonate to the mixture in 1 drop of water. The reaction mixture was stirred at 50 ° C. for 2.5 hours. Ethyl acetate was added and the organic phase was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was high vacuum dried and used in the next synthetic step without further purification. This gave 358.0 mg (84% of theory) of the title compound.

LC-MS (method _1): R t = 1.33 min; MS (ESIpos): m / z = 684 (M + H) +.

Intermediate C101
N- (3-aminopropyl) -N-{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} -2-hydroxyacetamide

  (1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropan-1-amine (intermediate C52) 150.0 mg ( 0.42 mmol) was first taken up in 2.0 mL dichloromethane, 29.2 mg (0.49 mmol) HOAc and 125.6 mg (0.59 mmol) sodium triacetoxyborohydride were added and the mixture was stirred at room temperature for 5 minutes . 3- (1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl) propanal 98.9 mg (0.49 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and the organic phase was washed twice with saturated sodium carbonate solution and once with saturated NaCl solution. After dehydration with magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified using silica gel (mobile phase: dichloromethane / methanol 100: 1). The solvent was distilled off under reduced pressure, and the residue was dried under high vacuum. This gave the compound 2- [3-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} amino. ) Propyl] -1H-isoindole-1,3 (2H) -dione 188.6 mg (74%).

LC-MS (Method 1): R _t = 1.00 min; MS (ESIpos): m / z = 541 [M + H] ⁺ .

  First, 2- [3-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} amino) Propyl] -1H-isoindole-1,3 (2H) -dione (171.2 mg, 0.32 mmol) was placed in 5.0 mL of dichloromethane, and 73.6 mg (0.73 mmol) of triethylamine was added. At 0 ° C., 94.9 mg (0.70 mmol) of acetoxyacetyl chloride was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and the organic phase was washed twice with saturated sodium bicarbonate solution and once with saturated NaCl solution. After dehydration with magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified using BiotageIsolera (silica gel, column 10 g SNAP, flow rate 12 mL / min, ethyl acetate / cyclohexane 1: 3). The solvent was distilled off under reduced pressure, and the residue was dried under high vacuum. This resulted in compound 2-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} [3- ( There were obtained 159.0 mg (77%) of ethyl 1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) propyl] amino) -2-oxoacetate.

LC-MS (method _1): R t = 1.35 min; MS (ESIpos): m / z = 642 [M + H] +.

  First, 2-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} [3- (1 , 3-Dioxo-1,3-dihydro-2H-isoindol-2-yl) propyl] amino) -2-oxoacetic acid ethyl ester (147.2 mg, 0.23 mmol) was added to ethanol (4.0 mL) and methanamine (40 % Aqueous solution) 356.2 mg (4.59 mmol) was added. The reaction mixture was stirred at 50 ° C. overnight. The solvent was distilled off under reduced pressure, and the residue was codistilled with toluene three times. The residue was purified using silica gel (mobile phase: dichloromethane / methanol 10: 1). The solvent was distilled off under reduced pressure, and the residue was dried under high vacuum. This gave 67.4 mg (63%) of the title compound.

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m / z = 470 [M + H] ⁺ .

Intermediate C102
tert-butyl {(2S) -4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} (Glycoloyl) amino] -1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetyl] amino} ethyl) amino] -1-oxobutane-2- Il} Carbamate

  (2S) -4-[{(1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino ] -2-[(tert-butoxycarbonyl) amino] butanoic acid (intermediate C5) 20.0 mg (32.54 μmol), N- (2-aminoethyl) -2- (2,5-dioxo-2,5 -Dihydro-1H-pyrrol-1-yl) acetamide trifluoroacetate (1: 1) (Intermediate L1) 10.1 mg (32.54 μmol) and HATU 18.6 mg (48.81 μmol) were dissolved in 2.5 mL of DMF. It was. 16.8 mg (23 μL, 130.15 μmol) of N, N-diisopropylethylamine was added and the mixture was stirred at room temperature for 1 hour. The mixture was purified directly by preparative HPLC (eluent: acetonitrile / water + 0.1% TFA, gradient 35: 65 → 95: 5) and then lyophilized to give 15 mg (58%) of the target compound. .

LC-MS (Method 1): R _t = 1.23 min; MS (EIpos): m / z = 794 [M + H] ⁺ .

Intermediate C103
tert-Butyl [(2S) -4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} (Glycoloyl) amino] -1-({2-[({3-[(2,2-dimethyl-4,12-dioxo-3,15,18,21,24-pentaoxa-5,11-diazahexa Cosan-26-yl) sulfanyl] -2,5-dioxopyrrolidin-1-yl} acetyl) amino] ethyl} amino) -1-oxobutan-2-yl] carbamate

  tert-butyl {(2S) -4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} (Glycoloyl) amino] -1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetyl] amino} ethyl) amino] -1-oxobutane-2- IL} carbamate (Intermediate C102) 15 mg (18.90 μmol) was dissolved in 500 μL of acetonitrile. Tert-butyl (7-oxo-21-sulfanyl-10,13,16,19-tetraoxa-6-azahenicos-1-yl) carbamate (intermediate L2) dissolved in 275 μL of phosphate buffered saline (pH = 7) ) 12.3 mg (26.45 μmol) was added to the mixture. The pH of the solution was adjusted to pH = 8 by adding a few drops of 1N sodium hydroxide solution. The mixture was stirred at room temperature for 30 minutes. The mixture was purified by preparative HPLC (eluent: acetonitrile / water + 0.1% TFA, gradient 35: 65 → 95: 5) and then lyophilized to give 20 mg (84%) of the target compound.

LC-MS (Method 1): R _t = 1.26 min; MS (EIpos): m / z = 1261 [M + H] ⁺ .

Intermediate C104
(2S) -4-[{(1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino ] -2-{[(Benzyloxy) carbonyl] amino} butanoic acid

  Intermediate C52 was reductively alkylated with benzyl- (2S) -2-{[(benzyloxy) carbonyl] amino} -4-oxobutanoate in the same manner as Intermediate C2. The secondary amino group was then acylated with 2-chloro-2-oxoethyl acetate. In the final step, the two ester groups were cleaved with a solution of 2M lithium hydroxide in methanol.

LC-MS (Method 1): R _t = 1.31 min; MS (ESIpos): m / z = 646 (M−H) ⁻ .

Intermediate C105
Benzyl N-{(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2 -Dimethylpropyl} (glycoloyl) amino] butanoyl} -β-alaninate trifluoroacetate (1: 1)

  200 mg of intermediate C58 was coupled with benzyl β-alaninate in DMF in the presence of HATU and N, N-diisopropylethylamine, then the Teoc protecting group was converted to 4 equivalents of chloride over 40 minutes with heating at 50 ° C. Cleavage using zinc / 2,2,2 trifluoroethanol. After adding 4 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _12): R t = 1.7 min; MS (ESIpos): m / z = 675 (M + H) +.

Intermediate C106
2- (Trimethylsilyl) ethyl N- [2-({(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole] -2-yl] -2,2-dimethylpropyl} (glycoyl) amino] butanoyl} amino) ethyl] -N2-{[2- (trimethylsilyl) ethoxy] carbonyl} -L-glutamate

  151 mg (0.23 mmol) of intermediate C104 was coupled with 128 mg (0.234 mmol) of intermediate L9 in DMF in the presence of HATU and N, N-diisopropylethylamine. The Z-protecting group was then cleaved by hydrogenation with 10% palladium / activated carbon under normal pressure.

  Yield: 30% of theoretical in 2 steps.

LC-MS (method _1): R t = 1.14 min; MS (ESIpos): m / z = 929 (M + H) +.

Intermediate C108
2- (trimethylsilyl) ethyl ^{N 6 - (N - {(} 2S) -2- amino--4 - [{(1R) -1- [1- benzyl-4- (2,5-difluorophenyl)-1H-pyrrole -2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] butanoyl} -β-alanyl) -N ² -{[2- (trimethylsilyl) ethoxy] carbonyl} -L-lysinate

103 mg (0.16 mmol) of intermediate C104 are prepared in the presence of EDC, HOBT and N, N-diisopropylethylamine in DMF in 2- (trimethylsilyl) ethyl N ⁶ -β-alanyl-N ² -{[2- ( Coupling with 110 mg (0.175 mmol) of trimethylsilyl) ethoxy] carbonyl} -L-lysinate (intermediate L11). The Z-protecting group was then cleaved by hydrogenation in DCM-methanol 1: 1 with 10% palladium / activated carbon under normal pressure. The title compound was obtained in a yield of 113 mg (75% over 2 steps).

LC-MS (method _1): R t = 1.17 min; MS (ESIpos): m / z = 957 (M + H) +.

Intermediate C109
Dibenzyl N-{(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2 -Dimethylpropyl} (glycolyl) amino] butanoyl} -β-alanyl-L-glutamate

  Intermediate C61 coupling with dibenzyl L-glutamate in DMF in the presence of HATU and N, N-diisopropylethylamine followed by 10 equivalents of zinc chloride in trifluoroethanol heated at 50 ° C. for 1 hour. Removal of the Teoc protecting group using / 2,2,2 trifluoroethanol gave the title compound.

LC-MS (method _1): R t = 1.09 min; MS (ESIpos): m / z = 894 (M + H) +.

Intermediate C110
N2-acetyl-N- [2-({(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole-2] -Yl] -2,2-dimethylpropyl} (glycolyl) amino] butanoyl} amino) ethyl] -N6- (tert-butoxycarbonyl) -L-lysineamide

  Coupling of intermediate C104 with intermediate L13 in DMF in the presence of HATU and N, N-diisopropylethylamine and subsequent hydrogenation in DCM-methanol 1: 1 with 10% palladium / activated carbon under normal pressure. Removal of the Z-protection by gave the title compound.

LC-MS (method _1): R t = 0.96 min; MS (ESIpos): m / z = 826 (M + H) +.

Intermediate C111
2- (trimethylsilyl) ethyl (3R, 4R) -3-[({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2, 2-Dimethylpropyl} amino) methyl] -4-fluoropyrrolidine-1-carboxylate trifluoroacetate (1: 1)

  (1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropan-1-amine (108 mg, 304 μmol) (Intermediate C52 ) In DCM (56.0 mL) and 4 × molecular sieves were added sodium triacetoxyborohydride (90.2 mg, 426 μmol). The mixture was stirred at room temperature for 15 minutes and 2- (trimethylsilyl) ethyl (3R, 4S) -3-fluoro-4-formylpyrrolidine-1-carboxylate (97.3 mg, 98% purity, 365 μmol) (reference: WO2014 / 151030A1) was added. The reaction mixture was stirred at room temperature for 3.5 hours and then diluted with DCM. The organic layer was washed with saturated sodium bicarbonate and water. The organic layer was dehydrated with sodium sulfate and evaporated. The residue was purified by preparative RP-HPLC to give 1.39 g (24% of theory) of the title compound.

LC-MS (method _1): R t = 1.15 min; MS (ESIpos): m / z = 600 (M + H) +.

Intermediate C112
2- (trimethylsilyl) ethyl (3R, 4R) -3-{[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2, 2-Dimethylpropyl} (chloroacetyl) amino] methyl} -4-fluoropyrrolidine-1-carboxylate

  2- (trimethylsilyl) ethyl (3R, 4R) -3-[({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2, 2-dimethylpropyl} amino) methyl] -4-fluoropyrrolidine-1-carboxylate trifluoroacetate (1: 1) (intermediate C111) in a solution of 692.8 mg (0.88 mmol) in DCM (8.7 mL) To molecular sieves 4 ２９, 295.0 mg (2.91 mmol) of triethylamine and 418.9 mg (3.71 mmol) of chloroacetyl chloride were added. The reaction mixture was stirred at room temperature for 2.5 hours and diluted with DCM. The organic layer was washed with saturated sodium bicarbonate and saturated ammonium chloride. The organic layer was dehydrated with sodium sulfate and evaporated. The residue was diluted with 8.7 mL of DCM, and 4 × molecular sieves, 295.0 mg (2.91 mmol) of triethylamine and 418.9 mg (3.71 mmol) of chloroacetyl chloride were added. The reaction mixture was stirred at room temperature for 3 hours and diluted with DCM. The organic layer was washed with saturated sodium bicarbonate and saturated ammonium chloride. The organic layer was dried over sodium sulfate and evaporated to give 691 mg (74% of theory) of the title compound, which was used in the next step without further purification.

LC-MS (method _1): R t = 1.78 min; MS (ESIpos): m / z = 676 (M + H) +.

Intermediate C113
S- [2-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} {[(3R, 4R) -4-Fluoro-1-{[2- (trimethylsilyl) ethoxy] carbonyl} pyrrolidin-3-yl] methyl} amino) -2-oxoethyl] -L-cysteine

  To a suspension of L-cysteine 203.6 mg (1.68 mmol) and sodium bicarbonate 201.7 mg (2.40 mmol) in water (0.95 mL), 2- (trimethylsilyl) ethyl (3R, 4R) -3- {[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (chloroacetyl) amino] methyl}- A solution of 170.0 mg (0.24 mmol) 4-fluoropyrrolidine-1-carboxylate (Intermediate C112) in isopropanol (9.5 mL) and 1,8-diazabicyclo (5.4.0) undec-7-ene 438 .5 mg (2.40 mmol) was added. The reaction mixture was stirred at 50 ° C. for 3 hours. The reaction mixture was diluted with ethyl acetate and the organic layer was washed with saturated sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and evaporated to give 152 mg (83% of theory) of the title compound, which was used in the next step without further purification.

LC-MS (method _1): R t = 1.26 min; MS (ESIpos): m / z = 762 (M + H) +.

Intermediate L1
Trifluoroacetic acid / N- (2-aminoethyl) -2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetamide (1: 1)

  The title compound is prepared by classical methods of peptide chemistry starting from commercially available (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetic acid and tert-butyl (2-aminoethyl) carbamate. Manufactured.

HPLC (Method 11): R _t = 0.19 min;
LC-MS (Method 1): R _t = 0.17 min; MS (ESIpos): m / z = 198 (M + H) ⁺ .

Intermediate L2
tert-Butyl (7-oxo-21-sulfanyl-10,13,16,19-tetraoxa-6-azahenicos-1-yl) carbamate

  356 mg (1.757 mmol) tert-butyl (5-aminopentyl) carbamate, 496 mg (1.757 mmol) 1-sulfanyl-3,6,9,12-tetraoxapentadecane-15-acid and 801 mg (2.108 mmol) HATU Was dissolved in 5.95 mL of DMF. The reaction mixture was cooled in an ice bath and 681 mg (920 μL, 5.272 mmol) of N, N-diisopropylethylamine was added. The mixture was stirred at room temperature for 4 hours and stored in a 4 ° C. refrigerator overnight. The mixture was purified directly by preparative HPLC (eluent: acetonitrile / water + 0.1% TFA, gradient 20: 80 → 80: 20) and then lyophilized to give 255 mg (29%) of the target compound. .

LC-MS (Method 1): R _t = 0.87 min; MS (EIpos): m / z = 467 [M + H] ⁺ .

Intermediate L3
N ² - acetyl ^-N 6 - [(benzyloxy) carbonyl] -L- lysine

Benzyl carbonochloridate Dating N ² - is reacted with acetyl -L- lysine, to give the intermediate.

LC-MS (method _1): R t = 0.71 min; MS (ESIpos): m / z = 323 (M + H) +.

Intermediate L4
N ² - acetyl ^-N 6 - {[2- (trimethylsilyl) ethoxy] carbonyl}-L-lysine

  This intermediate was obtained by reacting 1-({[2- (trimethylsilyl) ethoxy] carbonyl} oxy) pyrrolidine-2,5-dione with N2-acetyl-L-lysine.

LC-MS (method _1): R t = 0.87 min; MS (ESIpos): m / z = 333 (M + H) +.

Intermediate L5
9H-Fluoren-9-ylmethyl {(5S) -5-acetamido-6-[(2-aminoethyl) amino] -6-oxohexyl} carbamate trifluoroacetate (1: 1)

Commercially available ^{N 2} - acetyl ^-N 6 - [(9H-fluoren-9-ylmethoxy) carbonyl] -L- lysine is reacted with tert- butyl (2-aminoethyl) carbamate, then Boc group with trifluoroacetic acid Removal gave this intermediate.

LC-MS (method _13): R t = 0.87 min; MS (ESIpos): m / z = 453 (M + H) +.

Intermediate L6
2- (Trimethylsilyl) ethyl 3-{[(benzyloxy) carbonyl] amino} -D-alaninate trifluoroacetate (1: 1)

  The title compound was prepared using classical peptide chemistry starting from 3-{[(benzyloxy) carbonyl] amino} -N- (tert-butoxycarbonyl) -D-alanine. Esterification with 2- (trimethylsilyl) ethanol using EDC / DMAP followed by deprotection of the Boc group with TFA gave 405 mg (58% over two steps) of the title compound.

LC-MS (Method 1): R _t = 0.75 min; MS (ESIpos): m / z = 339 (M + H) ⁺ .

Intermediate L7
N ² - acetyl ^-N 6 - [(benzyloxy) carbonyl] -L- lysyl -L- alanyl -L- alanyl -L- aspartic

  The synthesis of the title compound was coupled to N-[(benzyloxy) carbonyl] -L-alanyl-L-alanine and tert-butyl L-aspartate in the presence of HATU and N, N-diisopropylethylamine in DMF. The Z-protecting group was removed by hydrogenation using 10% palladium / activated carbon in methanol. The resulting intermediate was coupled with intermediate L3 in DMF in the presence of HATU and N, N-diisopropylethylamine and, in the final step, the tert-butyl ester group was cleaved with trifluoroacetic acid / DCM.

LC-MS (method _1): R t = 0.59 min; MS (ESIpos): m / z = 579 (M + H) +.

Intermediate L8
N ² - acetyl ^-N 6 - {[2- (trimethylsilyl) ethoxy] carbonyl} -L- lysyl -L- alanyl -L- alanyl -L- aspartic

  The synthesis of the title compound was coupled to N-[(benzyloxy) carbonyl] -L-alanyl-L-alanine and tert-butyl L-aspartate in the presence of HATU and N, N-diisopropylethylamine in DMF. The Z-protecting group was then removed by hydrogenation in methanol with 10% palladium / activated carbon under normal pressure. The resulting intermediate was coupled with intermediate L4 in DMF in the presence of HATU and N, N-diisopropylethylamine, then both protecting groups were combined with 1% in 7.5% trifluoroacetic acid / DCM. Removed under stirring for hours. In the final step, the amino group was reprotected with 1-({[2- (trimethylsilyl) ethoxy] carbonyl} oxy) pyrrolidine-2,5-dione in the presence of N, N-diisopropylethylamine in DMF.

LC-MS (method _1): R t = 0.71 min; MS (ESIpos): m / z = 589 (M + H) +.

Intermediate L9
2,2-Dimethylpropanoic acid-2- (trimethylsilyl) ethyl-N- (2-aminoethyl) -N ² -{[2- (trimethylsilyl) ethoxy] carbonyl} -L-glutamate (1: 1)

  First, (4S) -5-tert-butoxy-4-[(tert-butoxycarbonyl) amino] -5-oxopentanoic acid is prepared in the presence of HATU and N, N-diisopropylethylamine in the presence of benzyl- (2-amino Coupling with ethyl) carbamate. Next, both the Boc group and the tert-butyl ester group were cleaved with trifluoroacetic acid. The amino group is then again protected by first reacting with trimethylsilyl) ethoxy] carbonyl} oxy) pyrrolidine-2,5-dione in DMF / water in the presence of N, N-diisopropylethylamine and then EDC. The carboxy group was esterified with 2- (trimethylsilyl) ethanol in DCM using / DMAP. In the final step, the Z-protecting group was removed by hydrogenation with 10% palladium / activated carbon under normal pressure to give the title compound, which was purified by HPLC.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m / z = 434 (M + H) ⁺ .

Intermediate L10
tert-Butyl N-[(benzyloxy) carbonyl] -L-alanyl-L-alanyl-L-aspartate

  Coupling of N-[(benzyloxy) carbonyl] -L-alanyl-L-alanine with tert-butyl L-aspartate in DMF in the presence of HATU and N, N-diisopropylethylamine followed by Removal of the tert-butyl ester group with trifluoroacetic acid in DCM gave the title compound.

LC-MS (Method 1): R _t = 0.5 min; MS (ESIpos): m / z = 409 (M + H) ⁺ .

Intermediate L11
2- (trimethylsilyl) ethyl ^N 6-.beta.-alanyl ^-N 2 - {[2- (trimethylsilyl) ethoxy] carbonyl}-L-lysinate

2- (Trimethylsilyl) ethyl N ² -[(benzyloxy) carbonyl] -L— of N- (tert-butoxycarbonyl) -β-alanine using HATU and N, N-diisopropylethylamine by classical methods of peptide chemistry. Starting from coupling with lysinate, removal of the Z-protecting group by hydrogenation with 10% palladium / activated carbon under normal pressure, 1-({[2- (trimethylsilyl) ethoxy] carbonyl} oxy) pyrrolidine-2, Introduction of the trimethylsilyl-ethyloxycarbonyl (Teoc) -protecting group with 5-dione and finally the mild removal of the Boc-protecting group by stirring for 45 minutes in a solution of 7.5% trifluoroacetic acid in dichloromethane. Was synthesized.

LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m / z = 462 (M + H) ⁺ .

Intermediate L12
Benzyl (2S) -5-({(5S) -5-acetamido-6-[(2-aminoethyl) amino] -6-oxohexyl} amino) -2-{[(benzyloxy) carbonyl] amino}- 5-oxopentanoate trifluoroacetate (1: 1)

  10% palladium / activated carbon under normal pressure starting from coupling of tert-butyl (2-aminoethyl) carbamate with intermediate L3 using HATU and N, N-diisopropylethylamine according to classical methods of peptide chemistry Removal of the Z-protecting group by hydrogenation in DCM / methanol 1: 1 with the resulting intermediate (4S) -5- (benzyloxy) -4- using HATU and N, N-diisopropylethylamine Removal of the Boc-protecting group by coupling with {[(benzyloxy) carbonyl] amino} -5-oxopentanoic acid and finally stirring for 1 hour in a solution of 25% trifluoroacetic acid in dichloroethane. Was synthesized.

LC-MS (method _1): R t = 0.66 min; MS (ESIpos): m / z = 584 (M + H) +.

Intermediate L13
Benzyl (2S) -5-({(5S) -5-acetamido-6-[(2-aminoethyl) amino] -6-oxohexyl} amino) -2-{[(benzyloxy) carbonyl] amino}- 5-oxopentanoate trifluoroacetate (1: 1)

Benzyl (2-aminoethyl) carbamate hydrochloride (1: 1) of N2-acetyl-N6- (tert-butoxycarbonyl) -L-lysine using HATU and N, N-diisopropylethylamine according to classical methods of peptide chemistry The title compound was then synthesized by removal of the Z-protecting group by hydrogenation in DCM / methanol 1: 1 with 10% palladium / activated carbon under normal pressure, starting from coupling with. LC-MS (Method 1): R _t = 0.43 min; MS (ESIpos): m / z = 331 (M + H) ⁺ .

Intermediate L14
N- (Pyridin-4-ylacetyl) -L-alanyl-L-alanyl-L-asparagine trifluoroacetate (1: 1)

  Using the classical method of peptide chemistry, commercially available tert-butyl L-alanyl-L-alaninate hydrochloride (1: 1) of pyridin-4-yl acetate hydrochloride (1: 1) using HATU and N, N-diisopropylethylamine Starting from the coupling with 1), the title compound was then synthesized by removing the tert-butyl ester with trifluoroacetic acid in DCM. The resulting intermediate was coupled with tert-butyl L-aspartate in the presence of HATU and N, N-diisopropylethylamine, and finally the tert-butyl ester was cleaved with trifluoroacetic acid / DCM.

LC-MS (method _1): R t = 0.15 min; MS (ESIpos): m / z = 394 (M + H) +.

Intermediate L15
2- (Trimethylsilyl) ethyl (3-oxopropyl) carbamate

  43. 4 mg (5.78 mmol) of 3-amino-1-propanol and 1.50 g (5.78 mmol) of 2- (trimethylsilyl) ethyl 2,5-dioxopyrrolidine-1-carboxylate were dissolved in 10.0 mL of dichloromethane, Triethylamine 585.3 mg (5.78 mmol) was added and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane and the organic phase was washed with water and saturated sodium bicarbonate solution and dried over magnesium sulfate. The solvent was distilled off under reduced pressure. The residue 2- (trimethylsilyl) ethyl (3-hydroxypropyl) carbamate (996.4 mg, 79% of theory) was dried under high vacuum and used in the next synthetic step without further purification.

  First, 807.0 mg (3.68 mmol) of 2- (trimethylsilyl) ethyl (3-hydroxypropyl) carbamate was added to 15.0 mL of chloroform and 0.05N potassium carbonate / 0.05N sodium bicarbonate solution (1: 1) 15. Put in 0 mL. Tetra-n-butylammonium chloride 102.2 mg (0.37 mmol), N-chlorosuccinimide 736.9 mg (5.52 mmol) and TEMPO 57.5 mg (0.37 mmol) were added and the reaction mixture was stirred vigorously at room temperature overnight. did. The reaction mixture was diluted with dichloromethane and the organic phase was washed with water and saturated NaCl solution. The organic phase was dehydrated with magnesium sulfate and the solvent was distilled off under reduced pressure. The residue was high vacuum dried and used in the next synthetic step without further purification (890.3 mg).

Intermediate L16
2,2-Dimethyl-4,11-dioxo-3,15,18,21,24-pentaoxa-5,12-diazaheptacosane-27-acid

  6-[(tert-Butoxycarbonyl) amino] hexanoic acid of methyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate in DMF in the presence of HATU and N, N-diisopropylethylamine And then saponification of the ester group with 5 equivalents of lithium hydroxide in THF: water (1: 1) at room temperature for 1 hour afforded the title compound.

LC-MS (Method 12): R _t = 1.25 min; MS (ESIpos): m / z = 479 [M + H] ⁺ .

Intermediate L17
2- (trimethylsilyl) ethyl 3 - ^{{[N 2} - acetyl ^-N 6 - (tert-butoxycarbonyl) -L- lysyl] amino}-D-alaninate

3-amino-N-[(benzyloxy) carbonyl] -D-alanine and 2,5-dioxopyrrolidin-1-yl N ² -acetyl-in DMF in the presence of N, N-diisopropylethylamine Coupling of N ^6- (tert-butoxycarbonyl) -L-liginate followed by carboxylic acid group 2- (trimethylsilyl) ethanol in acetonitrile in the presence of pyridine and 1,3-dicyclohexylcarbodiimide Coupling, followed by deprotection of the benzyloxycarbonyl group by hydrogenation in methanol in the presence of 10% Pd / C for 2 hours at room temperature gave the title compound.

LC-MS (method _1): R t = 0.70 min; MS (ESIpos): m / z = 475 (M + H) +.

Intermediate L81
Benzyl {2-[(2-aminoethyl) sulfonyl] ethyl} carbamate trifluoroacetate (1: 1)

  In the presence of N, N-diisopropylethylamine in DMF, 250 mg (1.11 mmol) of 2,2′-sulfonyldiethanamine was treated with 1-{[(benzyloxy) carbonyl] oxy} pyrrolidine-2,5-dione 92 Coupled with 3 mg (0.37 mmol). After HPLC purification, 70 mg (47% of theory) of the title compound were obtained.

LC-MS (method _12): R t = 0.64 min; MS (ESIpos): m / z = 257.11 (M + H) +.

Intermediate L108
N ² - ^{acetyl-N-(2-aminoethyl)} -N 6 - (tert- butoxycarbonyl) -L- lysine amide

N ² -acetyl-N ^6- (tert-butoxycarbonyl) -L-lysine and benzyl (2-aminoethyl) carbamate hydrochloride (1: 1) in DMF in the presence of HATU and N, N-diisopropylethylamine ) Followed by deprotection of the benzyloxycarbonyl group by hydrogenation in dichloromethane / methanol 1: 1 in the presence of 10% Pd / C for 1 hour at room temperature to give the title compound.

LC-MS (Method 1): R _t = 0.43 min; MS (ESIpos): m / z = 331 (M + H) ⁺ .

Intermediate F1
1-{[1- (2-{[2-({(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H] -Imidazol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] butanoyl} amino) ethyl] amino} -2-oxoethyl) -2,5-dioxopyrrolidin-3-yl] sulfanyl} -N -(5-Aminopentyl) -3,6,9,12-tetraoxapentadecane-15-amide dihydrochloride

  tert-Butyl [(2S) -4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-imidazol-2-yl] -2,2-dimethylpropyl} (Glycoloyl) amino] -1-({2-[({3-[(2,2-dimethyl-4,12-dioxo-3,15,18,21,24-pentaoxa-5,11-diazahexa Cosan-26-yl) sulfanyl] -2,5-dioxopyrrolidin-1-yl} acetyl) amino] ethyl} amino) -1-oxobutan-2-yl] carbamate (intermediate C103) 19 mg (15.07 μmol) Was dissolved in 300 μL of DCM. 113 μL of 4M hydrogen chloride in dioxane was added and the mixture was stirred at room temperature for 1 hour. An additional 100 μL of 4M hydrogen chloride in dioxane was added and the mixture was stirred again for 1 hour.

  The solvent was evaporated and the residue solidified by lyophilization from a mixture of acetonitrile and water to give 11 mg (64%) of the target compound.

LC-MS (Method 4): R _t = 5.86 min; MS (EIpos): m / z = 1060 [M + H] ⁺ .

Intermediate F2
N ² -acetyl-L-lysyl-L-valyl-N-{(1S) -3-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole- 2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] -1-carboxypropyl} -L-alaninamide trifluoroacetate (1: 1)

  Using classical methods known in peptide synthesis, methyl- (2S) -4-[(acetoxyacetyl) {(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole The title compound was prepared starting from -2-yl] -2,2-dimethylpropyl} amino] -2-({[2- (trimethylsilyl) ethoxy] carbonyl} amino) butanoate (intermediate C75). First, the Teoc group was cleaved with 6 equivalents of zinc chloride / 2,2,2 trifluoroethanol under heating at 50 ° C. for 2 hours in trifluoroethanol. The deprotected intermediate was then reacted with N-[(benzyloxy) carbonyl] -L-valyl-L-alanine in DMF in the presence of HATU and N, N-diisopropylethylamine. In the next step, the Z-protecting group was removed by hydrogenation with 10% palladium / activated carbon under normal pressure. The resulting intermediate was reacted with intermediate L3 in the presence of HATU and N, N-diisopropylethylamine in DMF. Subsequent ester cleavage with a solution of 2M lithium hydroxide in water / THF 2: 1 and final removal of the Z-protecting group by hydrogenation with 10% palladium / activated carbon under atmospheric pressure gave the title compound.

LC-MS (Method 1): R _t = 0.9 min; MS (ESIpos): m / z = 854 (M + H) ⁺ .

Intermediate F3
N ² - acetyl -L- lysyl -L- valyl -N- {3 - [{(1R ) -1- [1- benzyl-4- (2,5-difluorophenyl)-1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] propyl} -L-alaninamide trifluoroacetate (1: 1)

  N- (3-amino acid was coupled by coupling with benzyloxy) carbonyl] -L-valyl-L-alanine in the presence of HATU and N, N-diisopropylethylamine in DMF using known classical methods in peptide synthesis. Propyl) -N-{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} -2-hydroxyacetamide ( The title compound was synthesized starting from intermediate C101). In the next step, the Z-protecting group was removed by hydrogenation with 10% palladium / activated carbon under normal pressure. The resulting intermediate was reacted with intermediate L3 in the presence of HATU and N, N-diisopropylethylamine in DMF. Finally, the title compound was obtained by hydrogenation with 10% palladium / activated carbon under normal pressure.

LC-MS (method _1): R t = 0.90 min; MS (ESIpos): m / z = 810 (M + H) +.

Intermediate F4
3 - ^{[(N 2} - acetyl -L- lysyl) amino] -N - {(2S) -2- amino--4 - [{(1R) -1- [1- benzyl-4- (2,5-difluoro Phenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycolyl) amino] butanoyl} -D-alanine trifluoroacetate (1: 1)

  Intermediate C74 25 mg (0.026 mmol) was dissolved in 3.75 mL of DMF, and in the presence of HATU 12 mg (0.031 mmol) and 3 equivalents of N, N diisopropylethylamine, commercially available N2-acetyl-N6-[(9H- Fluoren-9-ylmethoxy) carbonyl] -L-lysine 13 mg (0.031 mmol) was coupled. In the second step, the Fmoc protecting group was cleaved with 100 equivalents of DABCO in 5 mL of DMF. Finally, the Teoc protecting group was cleaved with 6 equivalents of zinc chloride in trifluoroethanol with heating at 50 ° C. for 2 hours. After HPLC purification, 4.5 mg (20%) of the title compound was obtained.

LC-MS (method _1): R t = 0.76 min; MS (ESIpos): m / z = 770 (M + H) +.

Intermediate F5
N ² -acetyl-N- [2-({(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrole- 2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] butanoyl} amino) ethyl] -L-lysineamide trifluoroacetate (1: 1)

  (2S) -4-[{(1R) -1- [1-Benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino ] -2-{[(9H-Fluoren-9-ylmethoxy) carbonyl] amino} butanoic acid (intermediate C53) coupled to intermediate L5, followed by removal of the Fmoc protecting group by 100 equivalents of DABCO in DMF This gave the title compound.

LC-MS (method _12): R t = 1.11 min; MS (EIpos): m / z = 724.40 [M + H] +.

Intermediate F6
3 - ({15 - [( N 2 - acetyl -L- lysyl) amino] -4,7,10,13- tetra oxa pentadecane-1-Oil} amino) -N - {(2S) -2- Amino - 4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] butanoyl}- D-alanine trifluoroacetate (1: 1)

  In the first step, intermediate C74 was prepared from commercially available 9H-fluoren-9-ylmethyl {15-[(2,5-dioxopyrrolidin-1-yl) oxy]-in the presence of N, N diisopropylethylamine in DMF. 15-oxo-3,6,9,12-tetraoxapentadec-1-yl} carbamate was coupled. The Fmoc protecting group was then cleaved with 100 equivalents of DABCO in DMF and the resulting intermediate was coupled with L4 in the presence of 1.1 equivalents of HATU and 3 equivalents of N, N diisopropylethylamine in DMF. . Finally, the Teoc protecting group and trimethylsilylethyl ester were cleaved with 12 equivalents of zinc chloride in trifluoroethanol with heating at 50 ° C. for 1 hour. After HPLC purification, 2.3 mg of the title compound was obtained.

LC-MS (method _1): R t = 0.72 min; MS (EIpos): m / z = 1017 [M + H] +.

Intermediate F7
N ² - acetyl -L- lysyl -L- alanyl -L- alanyl ^{-N 1 - {(2S) -4} - [{(1R) -1- [1- benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] -1-[(2-carboxyethyl) amino] -1-oxobutan-2-yl} -L-aspartamide

  By coupling of intermediate C105 with intermediate L7 in the presence of HATU and N, N-diisopropylethylamine in DMF, followed by hydrogenation in DCM-methanol 1: 1 with 10% palladium / activated carbon. Simultaneous removal of the Z-protecting group and benzyl ester and HPLC purification gave the title compound.

LC-MS (method _1): R t = 0.82 min; MS (ESIpos): m / z = 1011 (M + H) +.

Intermediate F8
N ² - acetyl -L- lysyl -L- alanyl -L- alanyl ^{-N 1 - [(2S) -4} - [{(1R) -1- [1- benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] -1-{[2- (L-γ-glutamylamino) ethyl] amino} -1-oxobutan-2-yl] -L-aspartamide trifluoroacetate (1: 1)

  Coupling of intermediate C106 with intermediate L8 in the presence of HATU and N, N-diisopropylethylamine in DMF, followed by 10 equivalents of zinc chloride / 2,2 under heating at 50 ° C. for 6 hours Simultaneous removal of all protecting groups using trifluoroethanol gave the title compound. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _1): R t = 0.72 min; MS (ESIpos): m / z = 1111 (M + H) +.

Intermediate F9
N ² - acetyl -L- lysyl -L- alanyl -L- alanyl ^{-N 1 - {(2S) -1} - [(3 - {[(5S) -5- amino-5-carboxypentyl] amino} -3 -Oxopropyl) amino] -4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} ( Glycoroyl) amino] -1-oxobutan-2-yl} -L-aspartamide trifluoroacetate (1: 1)

  Coupling of intermediate C108 with intermediate L10 in the presence of HATU and N, N-diisopropylethylamine in DMF, then in DCM / methanol 1: 1 with 10% palladium / activated carbon under normal pressure Removal of the Z-protecting group by hydrogenation of, followed by coupling with intermediate L4 in the presence of HATU and N, N-diisopropylethylamine in DMF, finally 12 equivalents with heating at 50 ° C. for 2 hours Simultaneous removal of all protecting groups using a solution of zinc chloride / 2,2,2 trifluoroethanol gave the title compound. After adding 12 equivalents of EDTA, the product was purified by HPLC.

LC-MS (Method 12): R _t = 11.7 min; MS (ESIneg): m / z = 1137 (M−H) ⁻ .

Intermediate F10
N ² - acetyl -L- lysyl -L- alanyl -L- alanyl ^{-N 1 - {(2S) -4} - [{(1R) -1- [1- benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] -1-[(3-{[(1S) -1,3-dicarboxypropyl] amino} -3-oxopropyl Amino] -1-oxobutan-2-yl} -L-aspartamide trifluoroacetate (1: 1)

  Benzyl ester protection by coupling of intermediate C109 with intermediate L8 in the presence of HATU and N, N-diisopropylethylamine in DMF, followed by hydrogenation in methanol with 10% palladium / activated carbon under normal pressure Removal of the group and finally removal of the Teoc protecting group under 6 equivalents of zinc chloride / 2,2,2 trifluoroethanol under heating at 50 ° C. for 6 hours gave the title compound. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _12): R t = 1.31 min; MS (ESIpos): m / z = 1140 (M + H) +.

Intermediate F11
N ² - acetyl -L- lysyl -S- {2 - [(3- aminopropyl) {(1R) -1- [1- benzyl-4- (2,5-difluorophenyl)-1H-pyrrole-2 Yl] -2,2-dimethylpropyl} amino] -2-oxoethyl} -L-cysteine trifluoroacetate (1: 2)

S- (11-{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} -2,2-dimethyl -6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl) -L-cysteine trifluoroacetate (1: 1) (20.0 mg, 24.1 μmol) (intermediate) C71) and N ² -acetyl-N ^6- (tert-butoxycarbonyl) -L-lysine (9.02 mg, 31.3 μmol) in acetonitrile (2.0 mL) were added N, N-diisopropylethylamine (34 μL, 190 μmol) and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (19 μL, purity 0%, 31μmol) was added. The mixture was stirred at room temperature overnight and purified by preparative RP-HPLC (column: Reprosil 250 × 30; 10μ, flow rate: 50 mL / min, MeCN / water, 0.1% TFA). The solvent was distilled off under reduced pressure, and the residue was dried under high vacuum. Thereby, ^{N 2} - acetyl ^-N 6 - (tert-butoxycarbonyl) -L- lysyl -S- (11 - {(1R) -1- [1- benzyl-4- (2,5-difluorophenyl) - 1H-pyrrol-2-yl] -2,2-dimethylpropyl} -2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl) -L -5.2 mg (22% of theory) of cysteine were obtained. The resulting product contained about 15% of its epimer.

N ² - acetyl ^-N 6 - (tert-butoxycarbonyl) -L- lysyl -S- (11 - {(1R) -1- [1- benzyl-4- (2,5-difluorophenyl)-1H-pyrrole -2-yl] -2,2-dimethylpropyl} -2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl) -L-cysteine ( To a solution of 61.6 mg, 62.4 μmol) in 2,2,2-trifluoroethanol (6.0 mL) was added zinc chloride (51.0 mg, 374 μmol) and the mixture was stirred at 50 ° C. for 1 hour. Zinc chloride (51.0 mg, 374 μmol) was added and the mixture was stirred at 50 ° C. for 1 hour. The mixture was stirred with EDTA (218 mg, 748 μmol) for 5 min and purified by preparative RP-HPLC (column: Reprosil 250 × 30; 10 μ, flow rate: 50 mL / min, MeCN / water, 0.1% TFA). The solvent was distilled off under reduced pressure, and the residue was lyophilized. This gave 41.6 mg (69% of theory) of the title compound.

LC-MS (Method 12): R _t = 1.32 min; MS (ESIneg): m / z = 741 [M−H] ⁻ .

Intermediate F12
N- (6-aminohexanoyl) -S- [2-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2, 2-dimethylpropyl} {[(3S, 4R) -4-fluoropyrrolidin-3-yl] methyl} amino) -2-oxoethyl] -L-cysteine trifluoroacetate (1: 1)

  To a solution of 6-[(tert-butoxycarbonyl) amino] hexanoic acid (29.2 mg, 126 μmol) in DMF (1.4 mL) was added N, N-diisopropylethylamine (21 μL, 120 μmol) and HATU (46.2 mg, 122 μmol). ) Was added. The reaction mixture was stirred at room temperature for 10 minutes and S- [2-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2, 2-dimethylpropyl} {[(3R, 4R) -4-fluoro-1-{[2- (trimethylsilyl) ethoxy] carbonyl} pyrrolidin-3-yl] methyl} amino) -2-oxoethyl] -L-cysteine ( A solution of 37.0 mg, 48.6 μmol) (intermediate C107) in DMF (1.4 mL) was added. The reaction mixture was stirred at room temperature overnight. Water and DCM are added, the organic phase is separated, further washed with water, dried over magnesium sulfate, evaporated and S- [2-({(1R) -1- [1-benzyl-4- ( 2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} {[(3R, 4R) -4-fluoro-1-{[2- (trimethylsilyl) ethoxy] carbonyl} pyrrolidine There was obtained 49.7 mg (85% of theory) of -3-yl] methyl} amino) -2-oxoethyl] -N- {6-[(tert-butoxycarbonyl) amino] hexanoyl} -L-cysteine. Was used in the next step without further purification.

LC-MS (method _1): R t = 1.47 min; MS (ESIpos): m / z = 974 [M + H] +.

  S- [2-({(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} {[(3R, 4R) -4-fluoro-1-{[2- (trimethylsilyl) ethoxy] carbonyl} pyrrolidin-3-yl] methyl} amino) -2-oxoethyl] -N- {6-[(tert-butoxycarbonyl) amino] (45.2 mg, 332 μmol) was added to a solution of hexanoyl} -L-cysteine (49.7 mg, 81% purity, 41.4 μmol) in trifluoroethanol (3.5 mL) and the reaction mixture was stirred at 50 ° C. for 2 hours. Stir. EDTA (96.9 mg, 332 μmol) was added and the resulting mixture was stirred at room temperature for 15 minutes. Ethyl acetate was added and the organic phase was washed with water and brine, dried over magnesium sulfate and evaporated. The residue was purified by preparative RP-HPLC (MeCN / water, 0.1% TFA) to give 4.5 mg (13% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.79 min; MS (ESIpos): m / z = 730 [M + H] ⁺ .

Intermediate F13
N- (pyridin-4-ylacetyl) -L- alanyl -L- alanyl ^{-N 1 - {(2S) -1} - ({2 - [(N 2 - acetyl -L- lysyl) amino] ethyl} amino) - 4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] -1- Oxobutan-2-yl} -L-aspartamide trifluoroacetate (1: 1)

  Coupling of intermediate C110 with intermediate L14 in the presence of HATU and N, N-diisopropylethylamine in DMF, then 6 equivalents of zinc chloride / 2,2 with heating at 50 ° C. for 0.5 h Removal of the Boc group using, 2 trifluoroethanol gave the title compound. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _1): R t = 0.76 min; MS (ESIpos): m / z = 1101 (M + H) +.

Intermediate F14
N ² -acetyl-N- (2-{[2-({(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5-difluorophenyl)- 1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycoloyl) amino] butanoyl} amino) ethyl] sulfonyl} ethyl) -L-lysineamide trifluoroacetate (1: 2)

  First, intermediate L81 was coupled with intermediate C58 in the presence of HATU and N, N-diisopropylethylamine. The Z protecting group was then removed by hydrogenation in DCM / methanol 1: 1 with 10% palladium / activated carbon under normal pressure. The resulting intermediate was coupled with intermediate L4 in the presence of HATU and N, N-diisopropylethylamine.

  Finally, the Teoc protecting group was cleaved with 6 equivalents of zinc chloride / 2,2,2 trifluoroethanol with heating at 50 ° C. for 2 hours. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (Method 12): R _t = 0.7 min; MS (ESIpos): m / z = 818 (M + H) ⁺ .

Intermediate F15
N ² - Acetyl -N- {2 - [(3 - {[2 - ({(1R) -1- [1- benzyl-4- (2,5-difluorophenyl)-1H-pyrrol-2-yl] -2,2-dimethylpropyl} [pyrrolidin-3-ylmethyl] amino) -2-oxoethyl] sulfanyl} propanoyl) amino] ethyl} -L-lysineamide trifluoroacetate (1: 2) (isomer 1)

  Coupling of intermediate C95 with intermediate L108 in DMF in the presence of HATU and N, N-diisopropylethylamine, followed by 6 equivalents of zinc chloride / 2,2 under heating at 50 ° C. for 1 hour Removal of the Boc group using trifluoroethanol gave the title compound. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _1): R t = 0.74 min; MS (ESIpos): m / z = 796 (M + H) +.

Intermediate F16
N- (15-amino-4,7,10,13-tetraoxapentadecane-1-oil) -S- [2-({(1R) -1- [1-benzyl-4- (2,5-difluoro Phenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} [pyrrolidin-3-ylmethyl] amino) -2-oxoethyl] cysteine trifluoroacetate (1: 2) (isomer 1)

  2,2-Dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaeicosane-20-acid of intermediate C90 in the presence of HATU and N, N-diisopropylethylamine in DMF Coupling, followed by removal of the Boc group with 6 equivalents of zinc chloride / 2,2,2 trifluoroethanol under heating at 50 ° C. for 3 hours afforded the title compound. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _1): R t = 0.80 min; MS (ESIpos): m / z = 846 (M + H) +.

Intermediate F17
N- (22-amino-17-oxo-4,7,10,13-tetraoxa-16-azadocosane-1-oil) -S- {2-[(3-aminopropyl) {(1R) -1- [ 1-benzyl-4- (2,5-difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} amino] -2-oxoethyl} -L-cysteine trifluoroacetate (1: 2)

  Coupling of intermediate C71 with intermediate L16 in the presence of HATU and N, N-diisopropylethylamine in DMF, then 6 equivalents of zinc chloride / 2,2, heated at 50 ° C. for 1 hour. Removal of the protecting group using 2 trifluoroethanol gave the title compound. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _1): R t = 0.78 min; MS (ESIpos): m / z = 933 (M + H) +.

Intermediate F18
3 - ^{[(N 2} - acetyl -L- lysyl) amino] -N- [3 - ({2 - [(3- aminopropyl) {(1R) -1- [1- benzyl-4- (2,5 -Difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} amino] -2-oxoethyl} sulfanyl) propanoyl] -D-alanine trifluoroacetate (1: 2)

  Coupling of intermediate C69 with intermediate L17 in the presence of HATU and N, N-diisopropylethylamine in DMF, followed by 6 equivalents of zinc chloride / 2,2 with heating at 50 ° C. for 1 hour Removal of the protecting group using 2 trifluoroethanol gave the title compound. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _1): R t = 0.76 min; MS (ESIpos): m / z = 814 (M + H) +.

Intermediate F19
N ² - Acetyl -N- (2 - {[3 - ({2 - [(3- aminopropyl) {(1R) -1- [1- benzyl-4- (2,5-difluorophenyl)-1H- Pyrrol-2-yl] -2,2-dimethylpropyl} amino] -2-oxoethyl} sulfanyl) propanoyl] amino} ethyl) -L-lysine amide trifluoroacetate (1: 2)

  Coupling of intermediate C69 with intermediate L108 in the presence of HATU and N, N-diisopropylethylamine in DMF, then 6 equivalents of zinc chloride / 2,2, heated at 50 ° C. for 1 hour. Removal of the protecting group using 2 trifluoroethanol gave the title compound. After adding 6 equivalents of EDTA, the product was purified by HPLC.

LC-MS (method _1): R t = 0.79 min; MS (ESIpos): m / z = 770 (M + H) +.

B: Production of antibody / active compound complex (ADC) B-1. General methods for forming anti-TWEAKR antibodies Anti-TWEAKR antibodies were formed, for example, by screening phage display libraries for recombinant human TWEAKR SEQ ID NO: 138 and mouse TWEAKR SEQ ID NO: 137. In particular, the antibody TPP-2090 is an important example. The antibody thus obtained was reformatted into the human IgG1 format. The deglycosylated variant TPP-2090-HC-N297A was formed by introducing the mutation N297A into the heavy chain of TPP-2090 (Kabat numbering system for immunoglobulins). The deglycosylated variant TPP-2090-HC-N297Q was formed by introducing the mutation N297Q into the heavy chain of TPP-2090 (Kabat numbering system for immunoglobulins). These two antibodies were used in the working examples described herein (see also WO2015 / 189143A1 and WO2014 / 198817A1). Furthermore, antibodies that bind to TWEAKR are known to those skilled in the art, see for example WO2009 / 020933 (A2) or WO2009140177 (A2).

SEQ ID NO: 138 (polypeptide):
EQAPGTAPCSRGSSWSADLDKKCMDCASCRARPHSDFCLGCAAAPPAPFRLLWPRSDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWENGNGPEPNNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 137 (polypeptide):
EQAPGTSPCSSGSWSADSLKCMDCASCPARPHSDFCLGCCAAAPPAHFRLWPRSDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWENGNGPEPNNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
B-2. General Methods for Expression of Anti-TWEAKR Antibodies in Mammalian Cells Antibodies such as TPP-2090 and the deglycosylated mutants TPP-2090-HC-N297A, TPP-2090-HC-N297Q, trastuzumab-HC-N297A (TPP-7510 ), Trastuzumab-HC-N297Q (equivalent to TPP-7511), Tom et al. , Chapter 12 in Methods Express: Expression Systems, ed. Michelal R.C. Manufactured in a temporary mammalian cell culture according to the method described by Dyson and Yves Durocher, Scion Publishing Ltd, 2007 (AK—see Example 1).

B-3. General purification methods of antibodies from cell supernatants Antibodies such as TPP-2090 and deglycosylated mutants TPP-2090-HC-N297A, TPP-2090-HC-N297Q, trastuzumab-HC-N297A (equivalent to TPP-7510 ), Trastuzumab-HC-N297Q (equivalent to TPP-7511) was obtained from the cell culture supernatant. The cell supernatant was clarified by centrifugation of the cells. The cell supernatant was then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column. To that end, the column was equilibrated with DPBS pH 7.4 (Sigma / Aldrich), the cell supernatant was loaded, and the column was washed with about 10 column volumes of DPBS pH 7.4 + 500 mM sodium chloride. The antibody was eluted with 50 mM sodium acetate pH 3.5 + 500 mM sodium chloride and then further purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.

  The commercially available antibody cetuximab (trade name Erbitux) was purified from commercial products by standard chromatographic methods (Protein A, preparative SEC).

  The commercial antibody trastuzumab (trade name Herceptin) was purified from commercial products by standard chromatographic methods (Protein A, preparative SEC).

  Trastuzumab-HC-N297A (equivalent to TPP-7510) comprises the heavy chain represented by SEQ ID NO: 244. The light chain is identical to that of trastuzumab.

  Trastuzumab-HC-N297Q (equivalent to TPP-7511) comprises the heavy chain represented by SEQ ID NO: 245. The light chain is identical to that of trastuzumab.

  From a commercial product (trade name CIMAher), the antibody nimotuzumab was purified from the commercial product by standard chromatographic methods (protein A, preparative SEC).

  The antibody panitumumab was purified from the commercial product (trade name Vectibix) from the commercial product by standard chromatographic methods (protein A, preparative SEC).

B-4. General coupling method to glutamine
General coupling procedure for transglutaminase used in the examples below to obtain maximal DAR2:
For the reaction of the ADC working example, the following antibody was used (next name: antibody-HC-N297Z means an antibody with a substitution of N297 (Kabat numbering) by amino acid Z in both heavy chains of the antibody, The name TPP-XXXX-HC-Q295N-HC-N297Q replaces amino acid Q295 (Kabat numbering) with amino acid N in both heavy chains of the antibody and amino acid N297 with amino acid Q (Kabat numbering) in both heavy chains of the antibody. Means antibody to TPP-XXXX with a substitution of (Kabat numbering) The name of the antibody is referred to as the name (eg, trastuzumab) or TPP-XXXX (antibody with TPP-number XXXX).

AK _3a : TPP-2090-HC-N297A (equivalent to anti-TWEAKR antibody TPP-2658)
AK _3c : TPP-2090-HC-Q295N-HC-N297Q (equivalent to anti-TWEAKR antibody TPP-8825)
AK _3d : Trastuzumab-HC-N297A (equivalent to TPP-7510).

  Deglycosylation (glyco) variants of individual antibodies (HC-N297A or HC-Q295N-HC-N297Q) in a solution of 5 mg in DPBS pH 7.4 (c˜5 to 15 mg / mL) 20 μL (6 equivalents) of the solution (solution in 10 mM DMSO) was added. After 5 minutes incubation at 37 ° C., 50 μL (1.25 U) of an aqueous solution of recombinant microorganism (bacterial) transglutaminase (product number T001, from Zedira GmbH, Darmstadt, Germany) (25 U / mL) was added. The reaction mixture was incubated at 37 ° C. for 24 hours and then diluted with DPBS pH 7.4 to a volume of 2.5 mL. The ADC solution was purified by gel filtration on a PD10-column (Sephadex® G-25, GE Healthcare) equilibrated with DPBS-Buffer pH 7.4 (which was also used for elution). The ADC solution was then concentrated using an Amicon Ultracel-30K centrifuge (Millipore) and re-diluted to a volume of approximately 2.5 mL. Finally, 0.00625 μmol of b-transglutaminase blocker Zedira C100 in 12.5 μL of DPBS was added. About the obtained ADC solution, protein concentration was calculated | required by the method in each Example. The drug load was determined by the method described in Chapter B5. The ADC batch was characterized according to the method pointed out in the examples.

General procedure B for transglutaminase coupling used in the examples below to achieve maximum DAR4:
For the reaction of the ADC working example, the following antibodies were used (the following names are as used above):
AK _3b : TPP-2090-HC-N297Q (equivalent to anti-TWEAKR antibody TPP-5442)
AK _3e : Trastuzumab-HC-N297Q (equivalent to TPP-7511)
Deglycosylation of individual antibodies (aglyco) variant (HC-N297Q) in 5 mg solution in DPBS pH 7.4 (c ca. 5 to 15 mg / mL), 16 to 24 equivalents of a solution of individual precursor intermediate F (10 mM) Solution in DMSO). After 5 minutes incubation at 37 ° C., 400 μL (10 U) of an aqueous solution of recombinant microorganism (bacterial) transglutaminase (product number T001, from Zedira GmbH, Darmstadt, Germany) (25 U / mL) was added. The reaction mixture was incubated at 37 ° C. for 24 hours and then diluted with DPBS pH 7.4 to a volume of 2.5 mL. The ADC solution was purified by gel filtration on a PD10-column (Sephadex® G-25, GE Healthcare) equilibrated with DPBS-Buffer pH 7.4 (which was also used for elution). The ADC solution was then concentrated using an Amicon Ultracel-30K centrifuge (Millipore) and re-diluted to a volume of approximately 2.5 mL. Finally, 0.1 μmol of b-transglutaminase blocker Zedira C100 in 200 μL of DPBS was added. About the obtained ADC solution, protein concentration was calculated | required by the method in each Example. The drug load was determined by the method described in Chapter B7. The ADC batch was characterized according to the method pointed out in the examples.

General procedure for transglutaminase coupling on a larger scale used in the examples below to achieve maximum DAR2:
Deglycosylation (glyco) variants of individual antibodies (HC-N297A or HC-Q295N-HC-N297Q) in a solution of 30 mg in DPBS pH 7.4 (c˜5 to 15 mg / mL) with 6 equivalents of individual precursor intermediates A solution of body F (solution in 10 mM DMSO) was added. After 5 minutes incubation at 37 ° C., 200 μL (7.5 U) of an aqueous solution of recombinant microorganism (bacterial) transglutaminase (product number T001, from Zedira GmbH, Darmstadt, Germany) (25 U / mL) was added. The reaction mixture was incubated at 37 ° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) with DPBS 7.4 to remove small molecules and transglutaminase from the ADC, and finally using an Amicon Ultracel-30K centrifuge (Millipore). Concentrate to a final concentration of 5 to 25 mg / mL. The solution was sterile filtered.

  The individual protein concentrations described in the working examples of the ADC solution were determined. The drug load was determined by the method described in Chapter B5. The ADC batch was characterized according to the method pointed out in the examples.

General procedure for transglutaminase coupling on a larger scale used in the examples below to achieve maximum DAR4:
Deglycosylation (aglyco) variants of individual antibodies (HC-N297Q) 30 mg in DPBS pH 7.4 (c˜5 to 15 mg / mL) in 16 to 24 equivalents of individual precursor intermediate F solution (10 mM) Solution in DMSO). After incubation at 37 ° C. for 5 minutes, 2400 μL (60 U) of an aqueous solution of recombinant microorganism (bacterial) transglutaminase (product number T001, from Zedira GmbH, Darmstadt, Germany) (25 U / mL) was added. The reaction mixture was incubated at 37 ° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) with DPBS 7.4 to remove small molecules and transglutaminase from the ADC, and finally using an Amicon Ultracel-30K centrifuge (Millipore). Concentrate to a final concentration of 5 to 25 mg / mL. The solution was sterile filtered.

  The individual protein concentrations described in the working examples of the ADC solution were determined. The drug load was determined by the method described in Chapter B5. The ADC batch was characterized according to the method pointed out in the examples.

B-5. Antibody, measurement of chemotherapeutic load and measurement of binding site Trypsin digestion for protein identification in addition to molecular weight measurement after deglycosylation and / or denaturation, which is a tryptic peptide after denaturation, reduction and derivatization It confirms the identity of the protein via

  The poisoning body loading amount of the PBS buffer solution obtained from the complex described in the working example was determined as follows. Furthermore, it is believed that this approach can be used to identify coupling sites by detection of complexed trypsin peptides.

  Measurement of glutamate-coupled ADC loadings was performed by mass spectrometric measurement of the molecular weight of the individual complex species. Here, the samples were acidified and analyzed by mass spectrometry using an ESI-MicroTofQ system (Bruker Daltonik) after HPLC separation / desalting on a short C4 column (GromSil 300 Butyl-1 ST, 5 μm, 5 mm × 500 μm). All spectra with the signal in the TIC (total ion chromatogram) were added and the molecular weight of the different complex species was calculated based on MaxEnt deconvolution. Next, after signal integration of different species, the DAR (= drug / antibody ratio) was calculated by dividing the total number of poison-bearing weight-coupled species by the sum of the single weighted integration results for each chemical species.

  Furthermore, based on the species distribution, the homogeneity of the bTG coupling method (for DAR2, the percentage of D2> 85%) is, for example, by comparing the four different preparations of Example 5A in the table below. It is thought that it can be shown.

Table: Distribution comparison of four different productions of Example 5A (DAR2)

  Comparable uniform distribution can be obtained with other bTG coupling poison-linker constructs. This was shown by way of example with four different ADC compounds in the table below.

Table: Distribution of four different ADC examples (DAR2)

  Based on the N297Q substitution in the antibody, bTG based combinations with DAR4 results are possible. This ADC uniformity (percent D4> 70%) and mobility on different antibodies was shown by way of example for the three different ADC compounds in the table below.

Table: Distribution of two different ADC examples (DAR4)

  Alternatively, the amount of glutamate-linked complex loaded on the poison was determined by reverse-phase chromatography on reduced and denatured ADCs. A solution of guanidinium hydrochloride (GuHCl) (28.6 mg) and DL-dithiothreitol (DTT) (500 mM, 3 μL) was added to the ADC solution (1 mg / mL, 50 μL). The mixture was incubated at 55 ° C. for 1 hour and analyzed by HPLC.

  HPLC analysis was performed on an Agilent 1260 HPLC system with detection at 220 nm. Polymer Laboratories PLRP-S polymer reverse phase column (Cat. No. PL1912-3802) (2.1 × 150 mm, particle size 8 μm, 1000 μm) with the following gradient: 0 min, 31% B; 1 min, 31% B; 14 Min, 38% B; 16 min, 95% B at a flow rate of 1 mL / min. Mobile phase A consisted of 0.05% trifluoroacetic acid (TFA) / water and mobile phase B consisted of 0.05% trifluoroacetic acid / acetonitrile.

  The peaks detected were assigned by comparison of retention times of unconjugated antibody light chain (L0) and heavy chain (H0). The peaks detected exclusively in the complexed sample were assigned to heavy chains with one or two poisons (H1, H2).

  The average loading of the antibody with the poison is calculated from the peak area determined by integrating as the double of the sum of the HC loading and the LC loading, in which case the HC loading is the total heavy chain (HC ) The sum of the weighted integration results for the number of poisons in the peak divided by the sum of the results for the single weighted integration for the HC peaks. The sum is divided by the sum of the single weighted integration results of all LC peaks.

B-6. The ability of ADCs to bind the antigen binding check binder to the target molecule was examined after coupling occurred. One skilled in the art is familiar with the wide variety of methods used for this purpose, for example, the affinity of a complex is examined using ELISA techniques or surface plasmon resonance analysis (BIAcore ™ measurement). Can do. Conjugate concentration can be determined by one skilled in the art using, for example, general methods for antibody conjugates with proteins (Doronina et al .; Nature Biotechnol. 2003; 21: 778-784 and Polson et al., Blood 2007; see also 1102: 616-623).

Working example ADCs
The following examples were synthesized according to General Procedure C for Transglutaminase Coupling (see Chapter B-4).
Example 1A

  Here, DPBS pH 7.4 2250 μL, intermediate F1 solution 200 μL (10 mM solution in DMSO), and recombinant microbial (bacterial) transglutaminase solution (Product No. T001, Zedira GmbH, Darmstadt, Germany) (100 U / mL) 50 μL Was added to 2500 μL of a solution of antibody TPP-2090-HC-N297A (2 mg / mL). The reaction mixture was incubated at 37 ° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and transglutaminase from the ADC, and finally using an Amicon Ultracel-30K centrifuge (Millipore). Concentrated.

Protein concentration: 1.65 mg / mL
Drug / mAb ratio: 1.9.

Example 1A4

  Here, DPBS pH 7.4 450 μL, Intermediate F1 solution (solution in 10 mM DMSO) 40 μL, and recombinant microorganism (bacterial) transglutaminase solution (Product No. T001, Zedira GmbH, Darmstadt, Germany) (100 U / mL) 10 μL Was added to 500 μL of a solution of antibody TPP-2090-HC-N297Q (2 mg / mL). The reaction mixture was incubated at 37 ° C. for 24 hours and then directly analyzed for drug antibody ratio without further purification.

Protein concentration in the reaction mixture: 1 mg / mL
Drug / mAb ratio: 3.5.

Example 2A

  DPBS pH 7.4 50 μL, intermediate F2 solution (solution in 10 mM DMSO) 40 μL, and recombinant microbial (bacterial) transglutaminase solution (Product No. T001, Zedira GmbH, Darmstadt, Germany) (100 U / mL) 10 μL TPP-2090-HC-N297A solution (2 mg / mL) was added to 500 μL. The reaction mixture was incubated at 37 ° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and transglutaminase from the ADC, and finally using an Amicon Ultracel-30K centrifuge (Millipore). And concentrated.

Protein concentration: 1.26 mg / mL
Drug / mAb ratio: 1.6.

Example 3A

  Here, DPBS pH 7.4 450 μL, intermediate F3 solution (solution in 10 mM DMSO) 40 μL, and recombinant microorganism (bacterial) transglutaminase solution (Product No. T001, Zedira GmbH, Darmstadt, Germany) (100 U / mL) 10 μL Was added to 500 μL of a solution of antibody TPP-2090-HC-N297A (2 mg / mL). The reaction mixture was incubated at 37 ° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and transglutaminase from the ADC, and finally using an Amicon Ultracel-30K centrifuge (Millipore). And concentrated.

Protein concentration: 1.06 mg / mL
Drug / mAb ratio: 2.0.

Example 4A

  Here, DPBS pH 7.4 900 μL, intermediate F4 solution (solution in 10 mM DMSO) 80 μL, and recombinant microorganism (bacterial) transglutaminase solution (Product No. T001, Zedira GmbH, Darmstadt, Germany) (100 U / mL) 20 μL Was added to 1000 μL of a solution of antibody TPP-2090-HC-N297A (2 mg / mL). The reaction mixture was incubated at 37 ° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and transglutaminase from the ADC, and finally using an Amicon Ultracel-30K centrifuge (Millipore). And concentrated.

Protein concentration: 2.18 mg / mL
Drug / mAb ratio: 1.7.

Example 5A

  Here, DPBS pH 7.4 900 μL, intermediate F5 solution (solution in 10 mM DMSO) 80 μL, and recombinant microorganism (bacterial) transglutaminase solution (Product No. T001, Zedira GmbH, Darmstadt, Germany) (100 U / mL) 20 μL Was added to 1000 μL of a solution of antibody TPP-2090-HC-N297A (2 mg / mL). The reaction mixture was incubated at 37 ° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and transglutaminase from the ADC, and finally using an Amicon Ultracel-30K centrifuge (Millipore). And concentrated.

Protein concentration: 2.24 mg / mL
Drug / mAb ratio: 1.9.

Example 6A

  Here, DPBS pH 7.4 900 μL, intermediate F6 solution (solution in 10 mM DMSO) 80 μL, and recombinant microbial solution (bacteria) transglutaminase (Product No. T001, Zedira GmbH, Darmstadt, Germany) (100 U / mL) 20 μL Was added to 1000 μL of a solution of antibody TPP-2090-HC-N297A (2 / mg / mL). The reaction mixture was incubated at 37 ° C. for 24 hours, after which it was directly analyzed for drug antibody ratio without further purification.

Protein concentration in the reaction mixture: 2.22 mg / mL
Drug / mAb ratio: 2.0.

The following examples were synthesized according to General Procedure A or B for transglutaminase coupling on a 5 mg scale and according to Procedure C or D on a larger scale (see Chapter B-4).
Example 2A

Precursor: F2, General Procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.66 mg / mL
Drug / mAb ratio: 1.7.

Example 3A

Precursor: F3, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.71 mg / mL
Drug / mAb ratio: 1.8.

Example 4A

Precursor: F4, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.71 mg / mL
Drug / mAb ratio: 1.8.

Example 4A4

Precursor: F4, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 1.49 mg / mL
Drug / mAb ratio: 3.4.

Example 5A

Precursor: F5, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.35 mg / mL
Drug / mAb ratio: 2.0.

  This ADC coupling was also performed on a 30 mg scale according to General Procedure C in Chapter B4.

Protein concentration: 11.2 mg / mL
Drug / mAb ratio: 2.0.

Example 5A4

Precursor: F5, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 0.83 mg / mL
Drug / mAb ratio: 3.6.

  This ADC coupling was also performed on a 30 mg scale according to General Procedure D in Chapter B4.

Protein concentration: 10.0 mg / mL
Drug / mAb ratio: 3.8.

Example 5E

Precursor: F5, general procedure A, trastuzumab-HC-N297A (equivalent to TPP-7510)
Protein concentration: 2.44 mg / mL
Drug / mAb ratio: 1.6.

Example 5E4

Precursor: F5, General Procedure B, Trastuzumab-HC-N297Q (equivalent to TPP-7511)
Protein concentration: 2.85 mg / mL
Drug / mAb ratio: 3.6.

Example 6A

Precursor: F6, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.63 mg / mL
Drug / mAb ratio: 1.8.

  This ADC coupling was also performed on a 30 mg scale according to General Procedure C in Chapter B4.

Protein concentration: 13.2 mg / mL
Drug / mAb ratio: 1.9
Example 6A4

Precursor: F6, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 1.63 mg / mL
Drug / mAb ratio: 4.1.

  This ADC coupling was also performed on a 30 mg scale according to General Procedure D in Chapter B4.

Protein concentration: 11.8 mg / mL
Drug / mAb ratio: 3.7.

Example 7A

Precursor: F7, General Procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.66 mg / mL
Drug / mAb ratio: 1.9.

Example 8A

Precursor: F8, General Procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.68 mg / mL
Drug / mAb ratio: 1.9.

Example 9A

Precursor: F9, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.8 mg / mL
Drug / mAb ratio: 2.0.

Example 10A

Precursor: F10, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.73 mg / mL
Drug / mAb ratio: 1.8.

Example 10A4

Precursor: F10, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 1.76 mg / mL
Drug / mAb ratio: 3.9.

Example 10E4

Precursor: F10, General Procedure B, Trastuzumab-HC-N297Q (equivalent to TPP-7511)
Protein concentration: 1.56 mg / mL
Drug / mAb ratio: 3.9.

Example 11A

Precursor: F11, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.82 mg / mL
Drug / mAb ratio: 1.9.

Example 11A4

Precursor: F11, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 1.1 mg / mL
Drug / mAb ratio: 3.6.

Example 11E4

  Precursor: F11, general procedure B, trastuzumab-HC-N297Q (equivalent to TPP-7511).

Protein concentration: 1.75 mg / mL
Drug / mAb ratio: 3.8.

Example 12A

Precursor: F12, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.77 mg / mL
Drug / mAb ratio: 1.8.

Example 13A

Precursor: F13, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 2.11 mg / mL
Drug / mAb ratio: 1.8.

  This ADC coupling was also performed on a 30 mg scale according to General Procedure C in Chapter B4.

Protein concentration: 11.88 mg / mL
Drug / mAb ratio: 2.0.

Example 13A4

  Precursor: F13, general procedure D, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q).

  This ADC coupling was also performed on a 30 mg scale according to General Procedure C in Chapter B4.

Protein concentration: 12.0 mg / mL
Drug / mAb ratio: 3.8.

Example 14A

Precursor: F14, general procedure A, anti-TWEAKR antibody TPP-2658 (equivalent to TPP-2090-HC-N297A)
Protein concentration: 1.58 mg / mL
Drug / mAb ratio: 1.7.

Example 15A4

Precursor: F15, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 1.05 mg / mL
Drug / mAb ratio: 3.6.

Example 16A4

Precursor: F16, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 1.78 mg / mL
Drug / mAb ratio: 3.8.

Example 16E4

Precursor: F16, General Procedure B, Trastuzumab-HC-N297Q (equivalent to TPP-7511)
Protein concentration: 1.82 mg / mL
Drug / mAb ratio: 3.8.

Example 17A4

Precursor: F17, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 1.71 mg / mL
Drug / mAb ratio: 3.8.

Example 17E4

Precursor: F17, General Procedure B, Trastuzumab-HC-N297Q (equivalent to TPP-7511)
Protein concentration: 1.90 mg / mL
Drug / mAb ratio: 3.8.

Example 18A4

Precursor: F18, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 1.07 mg / mL
Drug / mAb ratio: 3.7.

Example 19A4

Precursor: F19, general procedure B, anti-TWEAKR antibody TPP-5442 (equivalent to TPP-2090-HC-N297Q)
Protein concentration: 0.92 mg / mL
Drug / mAb ratio: 3.3.

Metabolite example M100
N ⁶ -L-γ-glutamyl-N ² -acetyl-N- [2-({(2S) -2-amino-4-[{(1R) -1- [1-benzyl-4- (2,5) -Difluorophenyl) -1H-pyrrol-2-yl] -2,2-dimethylpropyl} (glycolyl) amino] butanoyl} amino) ethyl] -L-lysine amide trifluoroacetate (1: 1)

  First, intermediate C58 was coupled with intermediate L12 in the presence of HATU and N, N-diisopropylethylamine in DMF, and then the Teoc-protecting group was heated to 4 eq. After cleavage with zinc chloride / 2,2,2 trifluoroethanol and addition of 4 equivalents of EDTA, the product was purified by HPLC. Finally, the Z protecting group and benzyl ester were cleaved by hydrogenation in methanol with 10% palladium / activated carbon under normal pressure.

LC-MS (method _1): R t = 0.74 min; MS (ESIpos): m / z = 855 (M + H) +.

C: Evaluation of biological efficacy The biological activity of the compounds according to the invention can be demonstrated by the following evaluations.

a. C-1a: Measurement of ADC cytotoxic effect on TWEAKR The cytotoxic effect of anti-TWEAKR-ADC was analyzed using various cell lines.

  NCI-H292: human mucinous epidermoid lung cancer cells, ATCC-CRL-1848, standard medium: RPMI1640 (Biochrom; # FG1215, stable glutamine) + 10% FCS (Biochrom; # S0415), TWEAKR-positive, EGFR-positive.

  SK-HEP-1: human liver cancer cells, ATCC number HTB-52, standard medium: Earl salt + Glutamax I (Invitrogen 41090) + 10% heat-inactivated FCS (Fa. Gibco. Number 10500-064) -containing MEM; EGFR-positive, TWEAKR positive.

  LoVo: human colorectal cancer cells, ATCC number CCL-229, standard medium: Kain's + L-glutamine (Invitrogen 21127) + 10% heat-inactivated FCS (Fa. Gibco. Number 10500-064), TWEAKR-positive.

BxPC3: human pancreatic cancer cells, ATCC-CRL-1687, standard medium: RPMI1640 (Biochrom; # FG1215, stable glutamine) + 10% FCS (Biochrom; # S0415), TWEAKR-positive.
KPL4: human breast cancer cells, standard medium: RPMI 1640 + GlutaMAXI + 10% FBS, cell bank, Bayer Pharma AG (identity checked and confirmed on July 19, 2012 at DSMZ), Berlin, ERBB2-positive.

  Cells were cultured by standard methods as indicated by the American Tissue Culture Collection (ATCC) for individual cell lines.

MTT assay The test was performed by separating cells with Accutase in PBS (BiochromAG # L2143), pelleting, resuspending in medium, counting and seeding into 96 well culture plates with white bottom (Costar # 3610, LoVo: 1000 cells / well, SK-HEP-1: 1200 cells / well, NCI H292: 1500 cells / well in a total volume of 100 μL). The cells were then incubated at 37 ° C. and 5% carbon dioxide in an incubator. After 48 hours, antibody drug conjugates were added to 10 μL of medium (in triplicate) at concentrations of 10 ⁻⁵ M to 10 ⁻¹³ M and incubated at 37 ° C. and 5% carbon dioxide in an incubator. After 96 hours, proliferation was measured using the MTT assay (ATCC, Manassas, Virginia, USA, catalog number 30-1010K). After the selected incubation period, the cells were lysed overnight by adding MTT reagent, incubating with the cells for 4 hours, and then adding detergent. The resulting dye was detected at 570 nm (Infinite M1000pro, Fa. Tecan). Based on the measurement data, IC ₅₀ values were determined from DRC (dose response curve). The growth of cells not treated with the test substance but otherwise treated was defined as a 100% value. Data from selected examples are summarized in Table 1.

Table 1

Table 2

C-1b: Measurement of inhibition of kinesin spindle protein KSP / Eg5 The motor domain of human kinesin spindle protein KSP / Eg5 (from tebu-bio / Cytoskeleton Inc., No. 027EG01-XL) was added to 15 mM PIPES, pH 6.8 (5 mM MgCl ₂ and 10 mM DTT (from Sigma) with 50 μg / mL taxol (from Sigma, No. T7191-5MG) stabilized microtubule (bovine or pig, from tebu-bio / Cytoskeleton Inc) for 5 minutes at room temperature in Sigma) Incubate at a concentration of 10 nM. The freshly prepared mixture was aliquoted into 384 well MTP. Inhibitors and ATP (final concentration 500 μM from Sigma) at concentrations from 1.0 × 10 ⁻⁶ M to 1.0 × 10 ⁻¹³ M were added. Incubation was performed at room temperature for 2 hours. ATPase activity was detected by detecting inorganic phosphate formed using Malachite Green (from Biomol). After the reagent was added, the assay solution was incubated at room temperature for 50 minutes before detecting absorption at a wavelength of 620 nm. Monastrol (Fa. Sigma, M8515-1 mg) and ispinesive (from Adooq, A10486) were used as positive controls. Individual data for the dose-activity curve is an 8-fold measurement. IC ₅₀ values are the average of three independent experiments. The 100% control was a sample that was not treated with the inhibitor.

Table 2 below summarizes the IC ₅₀ values of representative examples derived from the described assay and corresponding cytotoxicity data (MTT assay).

Table 2

C-2: Internalization Assay Internalization is an important process that allows the specific and efficient provision of cytotoxic payloads in antigen-expressing cancer cells via antibody drug conjugates (ADC). This process is monitored via fluorescent labeling of specific TWEAKR antibodies and isotype control antibodies. First, a fluorescent dye is coupled to the lysine of the antibody. The conjugation is performed with a 2-fold molar excess of CypHer 5E mono NHS ester (batch 357392, GE Healthcare) at pH 8.3. After coupling, the reaction mixture was purified using gel chromatography (Zeba Spin Desalting Columns, 40K, Fa. Thermo Scientific, No. 87768; elution buffer: Dulbecco PBS, Fa. Sigma-Aldrich, No. D8537). Excess dye was removed and pH was adjusted. The resulting protein solution was concentrated (from VIVASPIN 500, Sartorius steady biotec). The measurement of the pigment loading amount of an antibody, spectrophotometric analysis (NanoDrop) and calculated in the following were carried out by _{(D: (A 280 -0.16A dye} ) ε dye: P = A dye ε protein). The dye loadings of the TWEAKR antibody and isotype control examined here were of the same order. In cell binding assays, it was confirmed that the binding did not result in a change in antibody affinity.

The labeled antibody was used in an internalization assay. Prior to this treatment, cells (2 × 10 ⁴ / well) were seeded in 100 μL of medium in 96-well MTP (thick, black, clear bottom No 4308776, from Applied Biosystems). After 18 hours incubation at 37 ° C./5% CO ₂ , the medium was changed and labeled TWEAKR antibody was added at different concentrations (10, 5, 2.5, 1, 0.1 μg / mL). The same treatment protocol was used for the labeled isotype control (negative control). The selected incubation times are 0 hours, 0.25 hours, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 6 hours and 24 hours. Fluorescence measurements were performed using an InCellanalyzer 1000 (from GE Healthcare). The kinetic evaluation was then performed by measuring the parameters granule count / cell and total granule strength / cell.

  After binding to TWEAKR, the TWEAKR antibody was examined for internalization ability. To that end, cells with different TWEAKR expression levels were selected. Target-mediated specific internalization was observed for the TWEAKR antibody, but the isotype control did not show any internalization.

  Commercially available antibodies (cetuximab, nimotuzumab, Herceptin) were similarly treated and internalized as described above using individual target expressing cells. For all antibodies, target-dependent internalization could be observed, but the isotype control did not show any internalization.

C-3: In vitro test to determine cell permeability The cell permeability of the substrate can be examined by an in vitro test in a flux assay using Caco-2 cells [M. D. Troutman and D.C. R. Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)]. In this regard, cells were cultured for 16 days from 15 cells on 24-well filter plates. To determine permeation, individual working examples were applied to cells with HEPES buffer solution (apicallyly (A) or basally (B)) and incubated for 2 hours. Samples were taken from the cis and trans compartments after 0 and 2 hours. The samples were separated by HPLC (Agilent 1200, Boeblingen, Germany) using a reverse phase column. The HPLC system was connected to a triple quadrupole mass spectrometer API4000 (Applied Biosystems Applera, Darmstadt, Germany) via the Turbo Ion Spray Interface. Permeability was evaluated based on P _app values calculated using the formula published by Schwab et al. [D. Schwab et al. , J .; Med. Chem. 46, 1716-1725 (2003)]. If P _{app (B-A) /} ratio of _P app (A-B) (discharge ratio) was> 2 or <0.5, it was classified as material is actively transported.

Of great importance for the toxins released within the cell are the permeability of B to A [P _app (BA)] and P _app (BA): P _app ( _AB ). Ratio (excretion ratio), the lower this permeability, the slower the active and passive transport of substances through the monolayer of Caco-2 cells. Furthermore, if the excretion ratio does not indicate active transport, the substance can remain longer in the cell after intracellular release. Therefore, the time available for interaction with the biochemical target (in this case kinesin spindle protein, KSP / Eg5) is also increased.

Table 3

C-4: In vitro test to determine substrate properties for P-glycoprotein (P-gp) Many tumor cells express transport proteins for drugs, which are very often cells Accompanying the development of resistance to growth inhibitors. Thus, it is believed that substances that are not substrates for such transport proteins, such as P-glycoprotein (P-gp) or BCRP, may exhibit improved activity profiles, for example.

Substrate properties of substances for P-gp (ABCB1) were determined by flux assay using LLC-PK1 cells (L-MDR1 cells) overexpressing P-gp [A. H. Schinkel et al. , J .; Clin. Invest. 96, 1698-1705 (1995)]. For this, LLC-PK1 cells or L-MDR1 cells were cultured in 96-well filter plates for 3-4 days. To measure permeability, individual test substances are singly or in the presence of an inhibitor (eg ivermectin or verapamil) in HEPES buffer solution, apically (A) or basally (basally). ) (B) Applied to cells and incubated for 2 hours. Samples were taken from the cis and trans compartments after 0 and 2 hours. The samples were separated by HPLC using a reverse phase column. The HPLC system was connected to a triple quadrupole mass spectrometer API3000 (Applied Biosystems Applera, Darmstadt, Germany) via the Turbo Ion Spray Interface. Permeability was evaluated based on P _app values calculated using the formula published by Schwab et al. [D. Schwab et al. , J .; Med. Chem. 46, 1716-1725 (2003)]. If P discharge ratio _{app (B-A) / P} app (A-B) was> 2, the material was classified as P-gp substrates.

  As a further criterion for assessment of P-gp substrate properties, the excretion ratio in L-MDR1 and LLC-PK1 cells or the excretion ratio in the presence or absence of an inhibitor can be compared. If these values differ by a factor greater than 2, the substance of interest is a P-gp substrate.

C-5: Pharmacokinetics
C5a: Description of confirmation method of ADC metabolites after internalization in vitro :
Internalization studies using immune complexes are performed to analyze intracellularly formed metabolites. For this, human lung tumor cells NCI H292 (3 × 10 ⁵ / well) are seeded in 6-well plates and incubated overnight (37 ° C., 5% CO ₂ ). The cells are treated with 10 μg / mL test ADC. Internalization is performed at 37 ° C. and 5% CO ₂ . At various time points (0, 4, 24, 48, 72 hours), take cell samples for further analysis. First, the supernatant (approximately 5 mL) is collected and stored at −80 ° C. after centrifugation (2 minutes, room temperature, 1000 rpm Heraeus Variofuge 3.0R). The cells are washed with PBS, separated using Accutase, and the number of cells is measured. After further washing, a predetermined number of cells (2 × 10 ⁵ ) were treated with 100 μL of lysis buffer (Mammalian Cell Lysis Kit (Sigma MCL1)) and continuously shaken in a Protein LoBind tube (Eppendorf catalog number 0030108.116). Incubate (Thermomixer, 15 min, 4 ° C., 650 rpm) After incubation, centrifuge the lysate (10 min, 4 ° C., 12000 g, Eppendorf 5415R) and collect the supernatant. Store at ° C. and analyze all samples as follows.

  Compounds in the culture supernatant or cell lysate are measured by high performance liquid chromatography (HPLC) coupled to a triple quadrupole mass spectrometer (MS) after protein precipitation with methanol or acetonitrile.

  For post-treatment of 50 μL of culture supernatant / cell lysate, 150 μL of precipitation reagent (typically acetonitrile) is added and the mixture is shaken for 10 seconds. The precipitation reagent contains an internal standard (ISTD) at a suitable concentration (usually in the range of 20 to 100 ng / mL). After centrifugation at 16000 g for 3 minutes, the supernatant is transferred to an autosampler vial, 500 μL of buffer appropriate for the mobile phase is added, and shaken again.

  The two matrix samples are then measured using an HPLC-coupled triple quadrupole mass spectrometer API 6500 from AB SCIEX Deutschland GmbH.

  For calibration, add 0.5 to 2000 μg / L of the concentrate to the plasma sample. The limit of detection (LOQ) is about 2 μg / L. The linear range is 2 to 1000 μg / L wide.

  To calibrate the tumor sample, add 0.5 to 200 μg / L of concentrate to the untreated tumor supernatant. The detection limit is 4 μg / L. The linear range is 4 to 200 μg / L wide.

  Quality controls to test for validity include 5 and 50 μg / L.

C5b: Confirmation of ADC metabolites in vivo After 3 to 30 mg / kg of different ADCs are administered intravenously, plasma and tumor concentrations of ADC and all resulting metabolites can be measured and clearance (CL) Pharmacokinetic parameters such as area under the curve (AUC) and half-life (t _1/2 ) can be calculated. Similarly, possible metabolite concentrations of ADC in plasma, tumors and tissues can be measured.

Quantitative analysis of the resulting metabolites After precipitation of the protein with methanol or acetonitrile, the compounds in plasma and tumor are measured by high performance liquid chromatography (HPLC) coupled to a triple quadrupole mass spectrometer (MS).

  For post-treatment of 50 μL of plasma, 250 μL of precipitation reagent (typically acetonitrile) is added and the mixture is shaken for 10 seconds. The precipitation reagent contains an internal standard (ISTD) at a suitable concentration (usually in the range of 20 to 100 ng / mL). After centrifugation at 16000 g for 3 minutes, the supernatant is transferred to an autosampler vial, 500 μL of buffer appropriate for the mobile phase is added, and shaken again.

  During tumor post-treatment, the latter is treated with 3 volumes of extraction buffer. The extraction buffer consisted of 50 mL tissue protein extraction reagent (Pierce, Rockford, IL), 2 pellets of complete-protease-inhibitor-cocktail (Roche Diagnostics GMBH, Mannheim, Germany) and phenylmethylsulfonyl fluoride at a final concentration of 1 mM. (Sigma, St. Louis, MO). Homogenize the sample twice for 20 minutes with a Tissueser II (Qiagen) at maximum stroke. Transfer 50 μL of the homogenate to an autosampler vial and add 150 μL of methanol containing ISTD. After centrifugation at 16000 g for 3 minutes, add 180 μL of a buffer suitable for the mobile phase to 10 μL of the supernatant, and shake again. The tumor sample is ready for measurement.

  The two matrix samples are then measured using an HPLC-linked triple quadrupole mass spectrometer API 6500 from AB SCIEX Deutschland GmbH.

  For calibration, add 0.5 to 2000 μg / L of the concentrate to the plasma sample. The limit of detection (LOQ) is about 2 μg / L. The linear range is 2 to 1000 μg / L wide.

  To calibrate the tumor sample, add 0.5 to 200 μg / L of concentrate to the untreated tumor supernatant. The detection limit is 5 μg / L. The linear range is 5 to 200 μg / L wide.

  Quality controls to test for validity include 5 and 50 μg / L, with an additional 500 μg / L in plasma.

Quantitative analysis of the antibodies used The antibody portion of the ADC was measured as the total IgG concentration in plasma samples and tumor lysates using a ligand binding assay (ELISA). Here, a sandwich ELISA method was used. This ELISA was qualified and validated for measurements on plasma and tumor samples. ELISA plates were coated with anti-human-goat IgG-Fc antibody. After incubation with the samples, the plates were washed and incubated with a detector complex of monkey anti-human IgG (H + L) antibody and horseradish peroxidase (HRP). After a further washing step, HRP substrate was added to the OPD and color development was monitored via absorption at 490 nm. Standard samples with known IgG concentrations were fitted using a four parameter equation. The unknown concentration was determined by interpolation within the range of the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ).

C-6: In vivo activity test The activity of the complex according to the present invention was examined using, for example, a xenograft model. The person skilled in the art is familiar with prior art methods by which the activity of the compounds according to the invention can be examined (for example WO 2005/081711; Polson et al., Cancer Res. 2009 Mar 15; 69 (6). : 2358-64). For this purpose, for example, tumor cell lines expressing the target molecule of the binder were transplanted into rodents (eg mice). The complexes according to the invention, isotype control complexes, control antibodies or isotonic saline were then administered to the implant animals. Administration was performed once or multiple times. After several days of incubation time, tumor size was measured by comparing the complex treated animals and the control group. Complex treated animals showed a relatively small tumor size.

Growth inhibition in mice / regression of experimental tumors
Human tumor cells expressing the antigen against the antibody drug conjugate are inoculated subcutaneously on the side of immunosuppressed mice, such as NMRi nude or SCID mice. One to ten million cells are removed from the cell culture, centrifuged, and resuspended in medium or medium / matrigel. Cell suspension is injected subcutaneously into mice.

The tumor grows within a few days. Treatment begins after the tumor is established with a tumor size of about 40 mm ² . In order to examine the effect on relatively large tumors, it is possible to start treatment only with a tumor size of 50 to 100 mm ² .

  Treatment with ADC is performed via the venous route to the tail vein of mice. ADC is administered in a volume of 5 mL / kg.

  The treatment protocol depends on the pharmacokinetics of the antibody. As standard, treatment is performed 3 consecutive times every 4 days. A protocol with a single treatment can be used for rapid assessment. However, treatment can be continued further or at a later time a second cycle of 3 treatment days can be performed.

  As a standard, 8 animals per treatment group are used. In addition to the group receiving the active substance, one group is treated as a control group with buffer only according to the same protocol.

  During the experiment, the tumor area is regularly measured in two dimensions (length / width) using a caliper. The tumor area is determined as length × width. The ratio of the mean tumor area of the treatment group to the control group is described as T / C area.

  After the treatment is complete, if all groups of the experiment are sacrificed simultaneously, the tumor can be removed and weighed. The ratio of the mean tumor weight of the treatment group to the control group is described as T / C weight.

D. Working Examples of Pharmaceutical Compositions The compounds according to the invention can be converted into the following pharmaceutical formulations.

IV solution:
Saturation of a compound according to the invention in a physiologically acceptable solvent (for example a formulation comprising glycine and sodium chloride in citrate buffer plus isotonic saline solution, D-PBS or polysorbate 80) Dissolve at a concentration below the solubility. The solution is sterile filtered and dispensed into sterile, pyrogen-free injection containers.

IV solution:
The compounds according to the invention can be converted into the stated administration forms. This is done in a manner known per se by means of “mixing” or “dissolving in” inert and non-toxic pharmaceutical suitable excipients (eg buffer substances, stabilizers, solubilizers, preservatives). be able to. For example, the following: amino acids (glycine, histidine, methionine, arginine, lysine, leucine, isoleucine, threonine, glutamic acid, phenylalanine and others), sugars and related compounds (glucose, saccharose, mannitol, trehalose, sucrose, mannose, lactose Sorbitol), glycerin, sodium, potassium, ammonium and calcium salts (eg sodium chloride, potassium chloride or disodium hydrogen phosphate and many others), acetate / acetate buffer systems, phosphate buffers, citrates Acid and citrate buffer systems, trometamol (TRIS and TRIS salts), polysorbates (eg, polysorbate 80 and polysorbate 20), poloxamers (eg, poloxamer 188 and polysorbate) Oxamer 171), macrogols (PEG derivatives, eg 3350), Triton X-100, EDTA salts, glutathione, albumins (eg human), urea, benzyl alcohol, phenol, chlorocresol, metacresol, benzalkonium chloride and Many other things may exist.

Lyophilizate for later intravenous, subcutaneous injection or intramuscular conversion:
Alternatively, the compounds of the invention can be converted into stable lyophilizates (perhaps with the aid of the above excipients) and suitable solvents (eg, water for injection, isotonic saline solution) prior to administration. Can be regenerated and administered.

Working Examples of Anti-TWEAKR Antibodies Unless otherwise described in detail herein, all examples were performed using standard methods known to those skilled in the art. The general methods of molecular biology of the examples below are described in Sambrook et al. , Molecular Cloning: a Laboratory Manual, Edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. , 1989, etc., can be carried out according to the method described in standard laboratory books.

AK Example 1: Antibody Production Using Antibody Library Dimers of human and mouse TWEAKR using a complete A human phage display library (Hoet RM et al, Nat Biotechnol 2005; 23 (3): 344-8) The TWEAKR-specific human monoclonal antibody of the present invention was isolated by protein panning (Hoogenboom HR, Nat Biotechnol 2005; 23 (3): 1105-16), which targets and fixes the Fc fusion extracellular domain.

Table AK-1: List of recombinant antigens used for antibody selection

Antigen was biotinylated using an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; catalog number 21347) according to the manufacturer's instructions and desalted using a Zeba desalting column (Pierce; catalog number 89889). Washed magnetic beads (DynaBeads) were incubated overnight at 4 ° C. with 200 nM biotinylated antigen and blocked for 1 hour at 4 ° C. with blocking buffer (PBS containing 3% BSA, 0.05% Tween-20). Blocked Fab phage library was added to blocked TWEAKR beads (DynaBeads Streptavidin-M280-Invitrogen 112-06D) and incubated at room temperature for 30 minutes. Stringent wash (3 times with blocking buffer and PBS containing 0.05% Tween-20 (150 mM NaCl; 8 mM Na ₂ HPO ₄ ; 1.5 mM KH ₂ PO ₄ ; pH adjusted to 7.4 to 7.6) 9)), then Fab phages that specifically bind to biotinylated TWEAKR beads (DynaBeads streptavidin-M280-Invitrogen 112-06D) were resuspended in PBS and Escherichia coli strain TG1 for amplification. Used directly for infection. In the second selection round, two mouse TWEAKRs (200 nM) were used to select for cross-reactive binders, and in the third selection round, the concentration of human TWEAKR was reduced (100 nM) and high affinity binders Increased selection pressure to.

  Eleven different Fab phages were identified and the corresponding antibody was cloned into a mammalian IgG expression vector, resulting in a missing CH2-CH3 domain that was not present in the soluble Fab. Tom et al. , Chapter 12 in Methods Express: Expression Systems ed. by Michele R. The resulting IgG was transiently expressed in mammalian cells according to the method described in Dyson and Yves Durocher, Scion Publishing Ltd, 2007. That is, an expression plasmid based on the CMV promoter was transfected into HEK293-6E cells and incubated in Fernbach bottles or Wave bags. Expression was performed in F17 medium (Invitrogen) at 37 ° C. for 5-6 days. Twenty-four hours after transfection, 1% Ultra-Low IgG FCS (Invitrogen) and 0.5 mM valproic acid (Sigma) were added as supplements. The antibody was purified by protein-A chromatography and further characterized by binding affinity to the soluble monomer TWEAKR using the AK-ELISA and BIAcore analysis described in Example 2.

Table AK-2: List of recombinant antigens used for affinity measurements

  To determine the cell binding properties of anti-TWEAKR antibodies, binding to many cell lines (HT29, HS68, HS578) was examined by flow cytometry. The cells were suspended in a dilution of antibody (5 μg / mL) in FACS buffer and incubated for 1 hour on ice. A second antibody (PE goat-anti-human IgG, Dianova # 109-115-098) was added. After 1 hour incubation on ice, cells were analyzed by flow cytometry using a FACS array (BD Biosciences).

NF-κB reporter gene assay was performed to evaluate agonist activity of all 11 identified antibodies (human IgG1). HEK293 cells were transiently transfected with the NF-κB reporter construct (BioCat, catalog number LR-0051-PA) using 293fectin according to the manufacturer's instructions. Transfected cells were seeded at 37 ° C., 5% CO ₂ in F17 medium (serum free; Invitrogen) in white polylysine-coated 384 well plates (BD). The next day, cells were stimulated with various concentrations of purified antibody for 6 hours prior to luciferase assay using standard methods.

Internalization was monitored via fluorescent labeling of anti-TWEAKR antibody (CypHer 5E mono NHS ester; GE Healthcare). Prior to treatment, HT29 cells were seeded (2 × 10 ⁴ / well) in 100 μL of medium in 96-well MTP plates (thick, black, clear bottom, No. 4308776, Applied Biosystems). After 18 hours incubation at 37 ° C./5% CO ₂ , the medium was changed and labeled anti-TWEAKR antibody was added at different concentrations (10, 5, 2.5, 1, 0.1 μg / mL). The incubation times selected were 0, 0.25, 0.5, 1, 1, 5, 2, 3, 6 and 24 hours. Fluorescence measurement was performed with InCell-Analyzer 1000 (GE Healthcare).

  The antibody with the highest in vitro activity (TPP-883) was selected for further activity and affinity maturation.

TPP-883
SEQ ID NO: 71
AQDIQMTQSPATLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GV
PSRFSGSGSGDFTFLTISSLQPEDFATYYC QQSYSSPGIT FGPGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLLNFYPPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 72
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDGYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWENGNGPEPNNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 71) and heavy chain (SEQ ID NO: 72) of TPP-883; both heavy and light chain CDRs are underlined.

Maturation was performed in the first round of mutation collection, followed by recombination of amino acids with the highest affinity and activity. To collect mutated NNK (N = AGCT, K = G or T), the following individual amino acid positions: S35, S36, Y37 and N39 at L1; A51, S53, S54, Q56 and S57 at CDR-L2; S92, Y93, S94, S95, G97 and I98 at CDR-L3; P31 at CDR-H1 Y32, P33, M34 and M35; Y50, S52, P53, S54, G56, K57 and H59 in CDR-H2; None in G99, G100, D101, G102, Y103, F104, D105 and Y106 in CDR-H3 Randomized. All individual NNK saturation mutagenesis library DNAs were cloned into mammalian IgG expression vectors for active maturation or phagemid vectors for affinity maturation. Affinity maturation was performed by phage panning. Washed magnetic beads (DynaBeads) were incubated with 10 nM, 1 nM, 100 pM or 10 pM biotinylated antigen overnight at 4 ° C. and blocking buffer (PBS containing 3% BSA, 0.05% Tween-20) for 1 hour at 4 ° C. Blocked. Blocked Fab phage libraries were added to blocked TWEAKR-DynaBeads at a 10,000-fold, 1000-fold or 100-fold excess compared to the theoretical library complexity and incubated for 30 minutes at room temperature. That means a total of 12 strategies were then performed (4 antigen concentration x 3 Fab phage titer). Fab that specifically binds to biotinylated TWEAKR DynaBeads (DynaBeads Streptavidin-M280-Invitrogen112-06D) after stringent washing (3 times with blocking buffer and 9 times with PBS containing 0.05% Tween-20) The phage was resuspended in PBS and used directly for infection of Escherichia coli strain TG1 for amplification. In selection round 2, the concentration of human TWEAKR-Fc was reduced (1 nM, 100 pM, 10 pM and 1 pM) and the same Fab phage titration was used for all 12 strategies (4.4 × 10 ¹¹ ). For the expression of soluble Fab, the phagemid vector was digested with MluI to remove the gene-III membrane anchor sequence required for Fab display on the phage and recombined the vector. 96 variants of each of the 12 selected pools were expressed as soluble Fabs and examined in an ELISA format. To this end, 2.5 nM biotinylated TWEAKR-Fc was antigen coated and soluble Fab binding was demonstrated using an anti-c-Myc antibody (Abcam ab62928). Seven single substitution variants (consecutive amino acid nomenclature) with improved binding to TWEAKR-Fc (SEQ ID NO: 138) are shown, CDR-L1 S36G, CDR-L2 A51Q and S57K, CDR-L3 S94T and G97F of CDR-H1, M35I of CDR-H1 and G102T of CDR-H3. For active maturation, HEK293 cells were transfected with NF-κB reporter (BioCat, catalog number LR-0051-PA). Transfected cells are seeded in white polylysine-coated 384-well plates (BD) in F17 medium (serum free; Invitrogen) and individual variants of the NNK diversified position antibody (human IgG1) library are transferred to mammals. Expressed transiently in cells. The next day, NF-κB reporter cells were stimulated with the expressed individual NNK antibody variants for 6 hours prior to luciferase assay using standard methods. One single substitution mutant with improved agonist activity was detected, which was CDR-H3 G102T. This mutant was also obtained by affinity maturation, which again showed the highest affinity promotion. After collecting mutations by affinity and activity screening, all seven preferred individual substitutions (library complexity: 128 variants) were recombined into a recombinant library. For this, oligonucleotides were synthesized to introduce selected mutations or corresponding wild type amino acids at each selected position. The library was established using successive rounds of overlap extension PCR. The final PCR product was ligated into a bacterial soluble Fab expression vector and 528 mutants were randomly selected for equilibrium ELISA screening with soluble Fab as described above (approximately 4-fold excess of the sample taken). Finally, 7 mutants were selected based on increased affinity compared to the best single substitution mutant G102T. These corresponding DNAs were cloned into mammalian IgG expression vectors and examined for functional activity in the NF-κB reporter cell assay. Finally, the resulting sequences were compared with human germline sequences to accommodate deviations that had no significant effect on affinity and potency. Antibodies with the following sequences were obtained by antibody library screening and affinity and / or activity maturation.

TPP-2090

SEQ ID NO: 1:
DIQMTQSPSSLSASVGDRVTITC RASQSISGYLN WYQQKPGKAPKLLIY QASSLQS GVPS
RFSGSGSGTDFLTTISSLQPEDFATYYC QQSYTSPFIT FGQGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLNLNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 2:
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMI WVRQAPGKGLEVS vs YISPSGGSTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequence of the light chain (SEQ ID NO: 1) and heavy chain (SEQ ID NO: 2) of TPP-2090;
Both heavy and light chain CDRs are underlined. Based on the sequence of TPP-2090, the deglycosylated antibodies TPP-2090-HC-N297A (containing mutations in heavy chain N297A (Kabat, EU numbering)) and TPP-2090-HC-N-297Q (heavy chain N297Q ( Kabat, EU numbering)) was formed by site-directed mutagenesis.

TPP-2090-HC-N297A
Light chain:
DIQMTQSPSSLSASVGDRVTITC RASQSISGYLN WYQQKPGKAPKLLIY QASSLQS GVPS
RFSGSGSGTDFLTTISSLQPEDFATYYC QQSYTSPFIT FGQGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLNLNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Heavy chain:
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMI WVRQAPGKGLEVS vs YISPSGGSTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKPREQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
TPP-2090-HC-N297Q
Light chain:
DIQMTQSPSSLSASVGDRVTITC RASQSISGYLN WYQQKPGKAPKLLIY QASSLQS GVPS
RFSGSGSGTDFLTTISSLQPEDFATYYC QQSYTSPFIT FGQGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLNLNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Heavy chain:
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMI WVRQAPGKGLEVS vs YISPSGGSTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEHVHNAKTTKREEQYQ
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
TPP-2149
SEQ ID NO: 11
DIQMTQSPATLSASVGDRVTITC RASQSISGYLN WYQQKPGKAPKLLIY QASSLQS GVPS
RFSGSGSGDFTFLTISSLQPEDFATYYC QQSYTSPFIT FGPGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLNLNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTL
TLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 12
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMI WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 11) and heavy chain (SEQ ID NO: 12) of TPP-2149; both heavy and light chain CDRs are underlined.

TPP-2093
SEQ ID NO: 21
DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY QASSLQS GVPS
RFSGSGSGTDFLTTISSLQPEDFATYYC QQSYTSPFIT FGQGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLNLNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 22
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPSGGSTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 21) and heavy chain (SEQ ID NO: 22) of TPP-2093; both heavy and light chain CDRs are underlined.

TPP-2148
SEQ ID NO: 31
DIQMTQSPATLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY QASSLQS GVPS
RFSGSGSGDFTFLTISSLQPEDFATYYC QQSYTSPFIT FGPGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLNLNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTL
TLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 32
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 31) and heavy chain (SEQ ID NO: 32) of TPP-2148; both heavy and light chain CDRs are underlined.

TPP-2084
SEQ ID NO: 41
DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPS
RFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSPGIT FGQGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLNLNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 42
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPSGGSTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 41) and heavy chain (SEQ ID NO: 42) of TPP-2084; both heavy and light chain CDRs are underlined.

TPP-2077
SEQ ID NO: 51
DIQMTQSPATLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPS
RFSGSGSGDFTFLTISSLQPEDFATYYC QQSYSSPGIT FGPGTKVEIIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLNLNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTL
TLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 52
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 51) and heavy chain (SEQ ID NO: 52) of TPP-2077; both heavy and light chain CDRs are underlined.

TPP-1538
SEQ ID NO: 61
AQDIQMTQSPATLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GV
PSRFSGSGSGDFTFLTISSLQPEDFATYYC QQSYSSPGIT FGPGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLLNFYPPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 62
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 61) and heavy chain (SEQ ID NO: 62) of TPP-1538; both heavy and light chain CDRs are underlined.

TPP-1854
SEQ ID NO: 81
AQDIQMTQSPATLSASVGDRVTITC RASQSISGYLN WYQQKPGKAPKLLIY NASSSLQS GV
PSRFSGSGSGDFTFLTISSLQPEDFATYYC QQSYTSPFIT FGPGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLLNFYPPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 82
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMI WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 81) and heavy chain (SEQ ID NO: 82) of TPP-1854; both heavy and light chain CDRs are underlined.

TPP-1853
SEQ ID NO: 91
AQDIQMTQSPATLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY NASSSLQS GV
PSRFSGSGSGDFTFLTISSLQPEDFATYYC QQSYTSPGIT FGPGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLLNFYPPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 92
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 91) and heavy chain (SEQ ID NO: 92) of TPP-1853; both heavy and light chain CDRs are underlined.

TPP-1857
SEQ ID NO: 101
AQDIQMTQSPATLSASVGDRVTITC RASQSISGYLN WYQQKPGKAPKLLIY NASSSLQS GV
PSRFSGSGSGDFTFLTISSLQPEDFATYYC QQSYTSPGIT FGPGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLLNFYPPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 102
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 101) and heavy chain (SEQ ID NO: 102) of TPP-1857; both heavy and light chain CDRs are underlined.

TPP-1858
SEQ ID NO: 111
AQDIQMTQSPATLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY NASSSLQS GV
PSRFSGSGSGDFTFLTISSLQPEDFATYYC QQSYTSPFIT FGPGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLLNFYPPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 112
EVQLLESGGGLVQPGGSLRLSCAASGFFTFS PYPMM WVRQAPGKGLEVS vs YISPGGKTHY
ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDY WGQGTLVTVSS
ASTKGPSVFFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
The amino acid sequences of the light chain (SEQ ID NO: 111) and heavy chain (SEQ ID NO: 112) of TPP-1858; both heavy and light chain CDRs are underlined.

Example 2: Biochemical characterization of antibodies Determination of binding affinity by Biacore analysis:
The binding affinity of the anti-TWEAKR antibody was determined using surface plasmon resonance analysis on a Biacore T100 instrument (GE Healthcare Biacore, Inc.). The antibody was immobilized on a CM5 sensor chip using an indirect capture reagent, anti-human IgG (Fc). Reagents from “Human Antibody Capture Kit” (BR-1008-39, GE Healthcare Biacore, Inc.) were used according to the manufacturer's instructions. Anti-TWEAKR antibody was injected at a concentration of 10 μg / mL for 10 seconds at 10 μL / min.

Table AK-3: List of recombinant antigens (TWEAKR) used for affinity measurements

Table AK-4: List of antibodies used for affinity measurement

Table AK-5: List of commercially available antibodies used for affinity measurement

Purified recombinant human TWEAKR protein (TPP) in various concentrations (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, 1.56 nM) in HEPES-EP buffer (GE Healthcare Biacore, Inc.) -2305, SEQ ID NO: 168) was injected over the immobilized anti-TWEAKR antibody at a flow rate of 60 μL / min over 3 minutes, with a dissociation time of 5 minutes. Sensorgrams were obtained after in-line reference cell correction and then subtraction of buffer samples. The dissociation constant (K _D ) was calculated based on the ratio of association (k _on ) and dissociation (k _off ) constants obtained by fitting the sensorgram using a 1: 1 primary binding model.

Table AK-6: TWEAKR protein (TPP-2305 (SEQ ID NO: 168)) using a monovalent _{K D} values of anti TWEAKR antibody was measured using a Biocore

Although the antibodies of the present invention bind to TWEAKR with moderate affinity (K _D 10 to 200 nM), several comparative antibodies (eg PDL-192 (TPP-1104), 136.1 (TPP-2194)) , 18.3.3 (TPP-2193), P4A8 (TPP-1324), P3G5 (TPP-2195), P2D3 (TPP-2196), ITEM-1, ITEM-4) have high affinity binding (from 0.7 3.7 nM) was confirmed. The sequences of the variable domains of the antibodies PDL-192, 136.1, 18.3.3, P4A8, P3G5 and P2D3 were obtained from the patent documents WO2009 / 020933 and WO2009 / 140177 and encoded the constant regions of human IgG1 and mouse IgG2. PDL-192 (TPP-1104), 136.1 (TPP-2194), 18.3.3 (TPP-2193), P4A8 (TPP-1324), P3G5 (TPP- 2195), P2D3 (TPP-2196) was obtained. The range of affinity measured in this study is in good agreement with published data; for PDL-192, 18.3.3 and 136.1, KD values of 5.5, 0.2 and 0.7 nM Is published (WO2009 / 020933); P4A8 is 2.6 nM (WO2009 / 140177). For comparison, the natural ligand TWEAK binds to TWEAKR with 2.4nM from _{the K D} value 0.8 (Immunity 2001 Nov; 15 ( 5):. 837-46; Biochem J. 2006 Jul 15; 397 (2) : 297-304; Arterioscler Thromb Vasc Biol. 2003 Apr 1; 23 (4): 594-600).

As a result, the antibodies of the present invention (TPP-883, TPP-1538, TPP-2077, TPP-2084, TPP-2148, TPP-2093, TPP-2149 and TPP-2090) have moderate affinity. (from _K D 10 200 nM) can be recorded to bind to TWEAKR in.

Characterization of the binding epitope of TPP-2090 using N-terminal and C-terminal truncation mutants of the TWEAKR extracellular domain:
The configuration of the cysteine-rich domain of TWEAKR (amino acids 34 to 68) of different species (Figure 1-configuration) shows that it is well conserved in the six species analyzed. Since PDL-192 binds depending on R56 (WO2009 / 020933: FIG. 2B), it does not bind to rat, pig and mouse TWEAKR. Since TPP-2090 binds depending on the conserved amino acid D47, it binds to all the species indicated.

  In an initial approach to characterize the binding epitope of the antibody, N-terminal and C-terminal truncated variants of the TWEAKR extracellular domain were formed and examined for their ability to bind to various anti-TWEAKR antibodies. The deletion of amino acids 28 to 33 at the N-terminus and amino acids 69 to 80 at the C-terminus resulted in a cysteine-rich domain with a disulfide bridge between Cys36-Cys49, Cys52-Cys67 and Cys55-Cys64. It remained intact (compare FIG. 2). Both constructs, total extracellular domain 28-80, including the N-terminus and C-terminus, and the truncated extracellular domain 34-68 were expressed and purified as the Fc fusion proteins TPP-2202 and TPP-2203, respectively.

  To analyze its binding, 1 μg / mL of the corresponding dimeric TWEAKR Fc construct was coated and 0.3 μg / mL and 0.08 μg / mL biotinylated IgG were used as soluble binding partners. Detection was performed using streptavidin-HRP and Amplex Red substrate. IgG was biotinylated using an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; catalog number 21347) and desalted using a Zeba desalting column (Pierce; catalog number 89889) according to the manufacturer's instructions. . At all concentrations using soluble ligand, the antibodies of the invention showed saturation binding to both constructs, whereas antibodies P4A8 (TPP-1324), P3G5 (TPP-2195) and Item-4 were in the full-length extracellular domain. Saturation binding was only shown for, and binding to N-terminal and C-terminal truncation constructs was worse (FIGS. 3 and 4). This indicates that the binding epitope of the antibody of the present invention is aligned with the cysteine-rich domain between amino acids 34-68. Monomers with a C-terminal deletion of amino acids 69 to 80 to analyze whether the N-terminal or C-terminal of the TWEAKR extracellular domain is required for P4A8 (TPP-1324) and P3G5 (TPP-2195) binding An extracellular domain was formed. The binding of P4A8 (TPP-1324) and P3G5 (TPP-2195) to the C-terminally cut TWEAKR extracellular domain is similarly worse, but the antibodies of the present invention show saturation binding (FIG. 5).

Table AK-9: List of recombinant antigens used in ELISA analysis for epitope profiling

Table AK-10: List of antibodies used in ELISA analysis for epitope profiling

  Thus, the binding epitopes of TPP-2090, TPP-2084, PDL-192 (TPP-1104) and 136.1 (TPP-2194) and the binding of P4A8 (TPP-1324) and P3G5 (TPP-2195) in the cysteine-rich domain The epitope is at least partially outside the cysteine-rich domain.

Effect of TWEAKR-Fc Mutein on Antibody Affinity In order to investigate the binding properties of the antibody of the present invention in more detail, it is suggested that TWEAKR is related to the activity of a known agonist antibody (WO2009 / 140177). Species muteins were examined. To this end, the full length extracellular domain (amino acids 28 to 80) with the following individual amino acid substitutions: T33Q; S40R; W42A; M50A; R56P; H60K; L65Q was expressed and purified as an Fc fusion protein.

Table AK-11: List of recombinant proteins used in ELISA analysis for mutein binding

  To obtain dose-response data, various TWEAKR-Fc muteins were coated at low concentrations (62 ng / mL) on 384-well Maxisorb ELISA plates and soluble in a series of 2-fold dilutions of biotinylated IgG starting at a concentration of 100 nM Used as a binding partner. Detection was performed using Streptavidin-HRP and Amplex Red. The examined IgGs are TPP-2090 and TPP-2084 of the present invention; PDL-192, 136.1 and 18.3.3 of WO2009 / 020933; P4A8 and P3G5 of WO2009 / 140177; and Nakayama et al [Biochem Biophys Res Com 306: 819-825].

Table AK-12: List of antibodies used in ELISA analysis for mutein binding

Table AK-13: List of commercially available antibodies used in ELISA for mutein binding

IgG was biotinylated using an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; catalog number 21347) according to the manufacturer's instructions and desalted using a Zeba desalting column (Pierce; catalog number 89889). . Dose - adapts the response data to determine the _{IC 50.} A tabulation was performed to show the results, with “−” indicating an IC ₅₀ greater than ₅₀ nM and “+” indicating an IC ₅₀ in the range of 1 to 150 pM.

Table AK-14: Effect of muteins on antibody binding

  As previously reported, ITEM-4 shows poor binding to H60K mutein [WO2009 / 140177: FIG. 23F] and PDL-192 shows poor binding to R56P mutein [WO2009 / 020933: FIG. 22B]. . In contrast to the published data, ITEM-1 shows worse binding to R56P and all antibodies show worsening to W42A [WO 2009/140177: FIG. 23E, FIG. 23F]. These differences can be explained by the method chosen.

  In contrast to ITEM-1, ITEM-4, PDL-192, 136.1 and 18.3.3, the antibodies of the present invention bind independently of all substitutions except W42A.

Alanine scans of cysteine-rich domains:
An alanine scan of the cysteine-rich domain (amino acids 34 to 68) was performed to determine the location of the binding site of the antibodies of the invention. FIG. 6 shows that N-terminal and C-terminal truncation mutants of the full-length extracellular domain of TWEAKR do not exacerbate the binding of the antibodies of the invention. Thus, the binding epitope is in the cysteine-rich domain. The following substitutions: S37A, R38A, S40A, S41A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A and L65A are TWEAKR (34-68) Fc Introduced into the construct.

Table AK-15: List of TWEAKR mutein constructs for alanine scanning of cysteine-rich domains

  These TWEAKR (34-68) Fc muteins were expressed in HEK293 cells. To obtain dose-response data, IgG was coated onto 384 well Maxisorp ELISA plates at a concentration of 1 μg / mL and serial 2-fold dilutions of the supernatant containing the TWEAKR mutein were used as soluble binding partners. Detection was performed using anti-HIS-HRP and Amplex Red. The IgGs examined were TPP-2090 of the present invention, PDL-192 of WO2009 / 020933 and P4A8 of WO2009 / 140177.

Table AK-16: List of antibodies used for alanine scanning of cysteine-rich domains

  To assess the relevance of the TWEAKR mutein for binding to various IgGs, correlation blots at certain mutein concentrations were made. For example, FIG. 6 shows a correlation blot for an 8-fold diluted supernatant of a TWEAKR expression broth, with PDL-192 (TPP-1104) on the X axis and TPP-2090 on the Y axis. The blot shows that the binding of TPP-2090 was aggravated by substitution D47A and that of PDL-192 (TPP-1104) was aggravated by substitution R56A. Binding to P4A8 (TPP-1324) is not shown for any construct, which is consistent with the results obtained above (FIG. 6). Thus, the P4A8 epitope is at least partially outside the cysteine-rich domain. The observed dependence on certain TWEAKR amino acids for antibody interactions correlates with the agonist activity measured for these antibodies. The natural ligand TWEAK shows effective activation of TWEAKR and binds depending on leucine 46 in the cysteine-rich domain of TWEAKR (Pellegrini et al, FEBS 280: 1818-1829). P4A8 exhibits very low agonist activity and interacts, at least in part, with domains outside the cysteine-rich domain of TWEAKR. PDL-192 exhibits moderate agonist activity and binds to a cysteine-rich domain depending on R56, but the TWEAK ligand site is reversed. Since TPP-2090 and TWEAK binding are dependent on D47 and L46, respectively, they bind to similar binding sites (FIG. 7).

  It can be concluded that the antibodies of the invention (eg TPP-2090) bind to TWEAKR in a manner dependent on D47.

  The observed dependence on certain TWEAKR amino acids for antibody interactions correlates with the agonist activity measured for these antibodies. The natural ligand TWEAK shows effective activation of TWEAKR and binds depending on leucine 46 in the cysteine-rich domain of TWEAKR (Pellegrini et al, FEBS 280: 1818-1829). P4A8 exhibits very low agonist activity and interacts, at least in part, with domains outside the cysteine-rich domain of TWEAKR. PDL-192 exhibits moderate agonist activity and binds to a cysteine-rich domain depending on R56, but the TWEAK ligand site is reversed. The antibody of the invention (Example TPP-2090) binds in a manner dependent on D47, TWEAK binds in a manner dependent on D46, and binds to a similar (but different) binding site (FIG. 7). Thus, the antibodies of the invention that exhibit strong agonist activity bind to a novel epitope (D47 dependent) for antibodies with very high agonist activity. An interesting point to note is that Michaelson et al. (See Michael JS et al, MAbs. 2011 Jul-Aug; 3 (4): 362-75, page 369, left column) It provides an explanation of the fact that agonist antibodies have weaker agonist activity than the natural ligand TWEAK. They conclude that potency reduction may be a function of the dimer binding interaction of the antibody with TWEAKR, and TWEAK probably enters a trimeric interaction. Thus, as a surprising result, the antibodies of the present invention have considerably higher agonist activity despite their dimeric interaction with TWEAKR. This surprising activity is related to the specific binding properties of the antibodies of the invention, ie the specific binding of TWEAKR to D47.

Claims (49)

A complex of a binder or derivative thereof with one or more active compound molecules, wherein the active compound molecule is a kinesin spindle protein inhibitor bound to the binder via a linker L, wherein the linker L is the binder 1 to 5 kinesin spindle protein inhibitors are bound to the linker L, and the kinesin spindle protein inhibitor bound to the linker L is represented by the following formula (IIa) And complexes, salts, solvates, solvate salts and epimers of the complex.

[Where:
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or Represents CF, X ₂ represents C, X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH or CF, X ₂ represents N, X ₃ represents C,
(Preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N);
R ¹ is -H, -L- # 1, -MOD or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
R ² represents —H, —L— # 1, —MOD, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents —H, —L— # 1, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
Z is ^{-H, -OY} 3, -SY 3, represents -NHY 3, -C (= O) -NY 1 Y 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ ;
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ⁴ represents R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) - or ^{_{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - group or a cathepsin cleavable group R _{^{21 - (C = O) (}} 0-1) - (P3) (1-2) -P2- represent,
R ²¹ is H, C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6- 10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - heterocyclic alkoxy group _(which, -NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (O) 2 -N ( alkyl _{2 -COOH, -C (= O) -NH} 2, -C (= O) -N ( _{alkyl) 2} or may be substituted one or more times by -OH,), - H or a group - _{(O) x} - represents a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids; when there are a plurality of amino acids P3, P3 can have the same or different amino acids as defined above;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ is —H, halogen (preferably —F or —Cl), —NH ₂ , —SO ₃ H, —COOH, —SH, C _1-4 -alkyl, halo-C _1-4 -alkyl, C _{1 Represents 4} -alkoxy, hydroxy-substituted C _1-4 -alkyl, —C (═O) —O— (C _1-4 -alkyl) or —OH, and a hydrogen atom of a secondary amino group in the pyrrolidine ring There _{^{R 21 -C (= O) -P3}} (0-2) -P2-NH-CH (CH 2 C (= O) -NH 2) -C (= O) -SIG- may be replaced by;
L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids; when there are a plurality of amino acids P3, P3 can have the same or different amino acids as defined above;
-SIG is a self-destructing group that gives a free secondary amine upon cleavage of the -C (= O) -SIG bond;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is -L- # 1, -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably C _1-10 -alkyl, C _6-10- Represents an aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, which is 1 to 3 —OH group, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 O-alkyl groups, 1 to 3 -SH group, 1 to 3 -S-alkyl group, 1 to 3 -O-C (= O) -alkyl group, 1 to 3 -O-C (= O) -NH-alkyl group 1 to 3 —NH C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, the one to three -S (= _{O) n} - alkyl group, one to three - S (═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 to 3 —NH ((CH ₂ CH ₂ O ) _1-20 H) group, optionally substituted by 1 to 3 —NH ₂ groups or 1 to 3 — (CH ₂ ) _0-3 Z groups,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
n represents 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ is _{-H, - (CH 2) 0-3} -CH (NH-C (= O) -CH 3) Z ', - (CH 2) 0-3 -CH (NH 2) Z' or - ( CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH,
R ⁵ is —L— # 1, H, —MOD, —NH ₂ , —NO ₂ , halogen (especially —F, —Cl, —Br), —CN, —CF ₃ , —OCF ₃ , —CH _{2. F,} -CH 2 _F, -SH or represents - _{(CH 2) 0-3} Z,
Z represents —H, —OY ³ , —SY ³ , halogen, —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} - represents alkynyl, _hydroxy, -NO _2, -NH 2, -COOH, or halogen (especially -F, -Cl, -Br), and - alkynyl, fluoro _{-C 2-10}
R ⁸ is C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _2-10 -alkynyl, fluoro-C _2-10- It represents (HZ ^2), - _{alkynyl, C 4-10} - cycloalkyl, fluoro _{-C 4-10} - cycloalkyl, - or _{- (CH 2)} 0-2
HZ ² represents a 4 to 7 membered heterocycle having 2 or less heteroatoms selected from the group consisting of N, O and S, each of these groups being —OH, —COOH or —NH ₂ or — Optionally substituted by L- # 1;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
One of the substituents R ¹ , R ² , R ³ , R ⁴ R ⁵ , R ⁸ and R ¹⁰ represents (or includes in the case of R ⁸ ) -L- # 1;
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ ;
In -MOD definition above _{^- (NR} 10) n ^- represents (G1) o -G2-G3,
R ¹⁰ represents H or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is —NH—C (═O) — or

, R ¹⁰ does not represent —NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y —, —C (═O) —, —CR ^x = N—O— may be interrupted one or more times,
Hydrocarbon chain containing either side _{chains, -NH-C (= O)} -NH 2, -COOH, -OH, -NH 2, NH-CN-NH 2, sulfonamide, sulfone, sulfoxide or sulfone acid May be replaced by
R ^y represents —H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NH—C (═O) —NH ₂ , — _{COOH, -OH, -NH 2, NH} -CN-NH 2, sulfonamide, sulfone, optionally substituted by a sulfoxide or sulfonic acid may,
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl;
G3 represents -H or -COOH;
-MOD preferably has at least one -COOH group. ] The complex according to claim 1, wherein X ₁ represents CH, X ₂ represents C, and X ₃ represents N. The complex according to claim 1 or 2, wherein the substituent R ¹ or the substituent R ³ represents -L- # 1. A complex of an active compound molecule that is a kinesin spindle protein inhibitor bound to a binder via a linker L and a binder or a derivative thereof, wherein the linker L is bound to a glutamine side chain of the binder, Complexes in which the kinesin spindle protein inhibitor has the following substructure, and salts, solvates, solvate salts and epimers thereof:

[Where:
#A represents binding to the rest of the molecule;
R ^1a represents —H, —MOD, or — (CH ₂ ) _0-3 Z;
-MOD is represented as defined above,
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —SO ₃ H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C═O—NH—CHY). ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ Represents;
R ^2a represents —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ Represents
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl;
R ^4a represents H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents —NHC (═O) —NH ₂ which may be substituted by linear or branched C _1-6 -alkyl, or aryl or benzyl which may be substituted by —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl,
Or R ^4a is R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P ₂ —NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O ) - group or cathepsin-cleavable group or a group ^R 21 of _{- (C = O) (0-1} ) - (P3) (0-2) -P2- represent,
R ²¹ represents C _{1-10 -alkyl-} , C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _6-10. - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy -, C _5-10 -heteroalkoxy-, C _1-10 -alkyl-O—C _{6-10 -aryloxy-} , represents a C _5-10 -heterocyclic alkoxy group, which represents —NH ₂ , —NH-alkyl, —N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -SO 2 -N ( _{alkyl) 2} , May be substituted one or more times by —COOH, —C (═O) —NH ₂ , —C (═O) —N (alkyl) ₂ or —OH; or it may be —H or a group — (O) _x - represents a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 10,
R ²² is, -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent N An alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
Or R ^2a and R ^4a together represent —CH ₂ —CHR ¹⁰ — (with formation of a pyrrolidine ring) or —CHR ¹⁰ —CH ₂ —,
R ¹⁰ represents —H, halogen (preferably —F), —NH ₂ , —COOH, —SO ₃ H, —SH or —OH;
The kinesin spindle protein inhibitor is linked to the linker by substitution of hydrogen atoms at R ^1a , R ^2a , R ^4a or at the pyrrolidine ring formed by R ^2a and R ^4a . ] The complex according to claim 4, wherein the kinesin spindle protein inhibitor is represented by the general formula (Ia), and salts, solvates, solvate salts and epimers thereof.

[Where:
R ^1a represents —H, —MOD or — (CH ₂ ) _0-3 Z;
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —SO ₃ H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH. -CHY ⁴⁾ represents the _1-3 COOH,
W represents -H or -OH;
Y ⁴ represents a linear or _branched C _1-6 -alkyl which may be substituted by —NH—C (═O) —NH ₂ , an aryl which may be substituted by —NH ₂ or Represents benzyl;
R ^2a represents —H, —MOD, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl;
R ^4a represents —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NHC (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . ,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl,
Or R ^4a is R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P ₂ —NH—CH (CH ₂ C— (C═O) —NH ₂ ) —C ( = O) -, or _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - group or, cathepsin cleavable group, or a group of the formula _{^{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2- represent,
R ²¹ represents —H, C _1-10 -alkyl-, C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C. _6-10 - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy _{-, C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl , -N _{(alkyl) 2, -NH-C (=} O) - alkyl, -N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -SO 2 NH} 2, -S (= O) ₂ -N (A _{Kill) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _alkyl) may be substituted one or more times with ₂ or -OH; or -H or a group -O _x - represents a _{(CH 2 CH 2 O) y} -R 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is, -H, - alkyl (preferably _C 1 _{- 12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent N An alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
Or R ^2a and R ^4a together represent —CH ₂ —CHR ¹⁰ — (with formation of a pyrrolidine ring) or —CHR ¹⁰ —CH ₂ —,
R ¹⁰ represents H, halogen (preferably F), —NH ₂ , —COOH, —SO ₃ H, —SH or —OH, and the hydrogen atom of the secondary amino group in the pyrrolidine ring is R ²¹ —C ( _{= O) -P3 (0-2) -P2} -NH-CH (CH 2 C (= O) NH 2) -C (= O) -SIG- it may be replaced by;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent N An alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
-SIG is a self-destructing group that provides a free secondary amine upon cleavage of the -C (= O) -SIG bond;
R ^3a is —MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl or heterocyclic alkyl group, preferably C _1-10 -alkyl, C _6-10 -aryl or C _{6- 10} -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group, which represents 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 O-alkyl groups, 1 to 3 —SH groups, To 3 -S-alkyl groups, 1 to 3 -O-C (= O) -alkyl groups, 1 to 3 -O-C (= O) -NH-alkyl groups, 1 to 3 -NH-C = O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - alkyl group, one to three -S ( ═O) ₂ —NH-alkyl group, 1 to 3 —NH-alkyl group, 1 to 3 —N (alkyl) ₂ group, 1 to 3 —NH ₂ group or 1 to 3 — (CH ₂ ) _0-3 optionally substituted by a Z group,
Z represents —H, halogen, —OY ³ , —SY ³ , —NHY ³ , —C (═O) —NY ¹ Y ² or —C (═O) —OY ³ ;
-MOD is represented as defined above,
n is 0, 1 or 2;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NH—C (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) _0-3 Z ′
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ^8a represents C _1-10 -alkyl,
HZ represents a monocyclic or bicyclic heterocycle, which is one or more selected from the group consisting of halogen, C _1-10 -alkyl group, C _6-10 -aryl group and C _6-10 -aralkyl group Which may be substituted by a substituent, which may be substituted by a halogen;
-MOD as defined ^above, _{- (NR} 10) n - represents (G1) o -G2-G3,
R ¹⁰ represents —H; halogen or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) —, —C (═O) —NH—, or

(G1 is —NHC (═O) — or

R ¹⁰ does not represent NH ₂ . );
n is 0 or 1;
o is 0 or 1;
G2 represents a straight-chain and / or branched hydrocarbon group, which has 1 to 10 carbon atoms and contains the groups -O-, -S-, -S (= O)-, -S (= O ) _2- , -NR ^y- , -NR ^y C (= O)-, -C (= O) -NR ^y- , -NR ^y NR ^y- , -S (= O) ₂ -NR ^y NR ^y- , —C (═O) —NR ^y NR ^y —, —C (═O) —, —CR ^x = N—O— may be interrupted one or more times,
The hydrocarbon group containing any side chain is replaced by —NHC (═O) —NH ₂ , —COOH, —OH, —NH ₂ , —NH—CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid. May have been
R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which is —NHC (═O) —NH ₂ , —COOH, _{-OH, -NH 2, NH-CN} -NH 2, sulfonamide, sulfone, optionally substituted by a sulfoxide or sulfonic acid may,
R ^x represents —H, C ₁ -C ₃ -alkyl or phenyl),
G3 represents -H or -COOH;
-MOD preferably has at least one group -COOH. ] The linker L is bound to the glutamine side chain of the binder, 1 to 5 kinesin spindle protein inhibitors are bound to the linker L, and the active compound molecular linker is represented by the general formula (II) 10. Complexes according to one or more of the preceding claims, and salts, solvates, solvate salts and epimers thereof.

[Where:
X ₁ represents N, X ₂ represents N, X ₃ represents C; or X ₁ represents N, X ₂ represents C, X ₃ represents N; or X ₁ represents CH or CF X ₂ represents C and X ₃ represents N; or X ₁ represents NH, X ₂ represents C, X ₃ represents C; or X ₁ represents CH and X ₂ represents N, X ₃ represents C,

R ¹ is -H, -MOD, -L- # 1 or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² are independently of each other —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ (eg, — (CH ₂ ) _0-3 Z ′. ) Or —CH (CH ₂ W) Z ′,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —NH ₂ , —SO ₃ H, —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—. CHY ⁴ ) _1-3 represents COOH,
W represents -H or -OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Representation;
R ² is -L- # 1, H, -MOD, -C (= O) -CHY 4 -NHY 5 or represents - _{(CH 2) 0-3} Z,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents linear or branched C _1-6 -alkyl;
R ⁴ represents —L— # 1, —H, —C (═O) —CHY ⁴ —NHY ⁵ or — (CH ₂ ) _0-3 Z;
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
Z is ^{-H, -OY} 3, -SY 3, represents -NHY 3, -C (= O) -NY 1 Y 2 or -C (= O) -OY ^3,
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted with —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted with —NH ₂ . Represent,
Y ⁵ represents —H or —C (═O) —CHY ⁶ —NH ₂ ;
Y ⁶ represents a linear or branched C _1-6 -alkyl,
Or R ⁴ represents R ²¹ — (C═O) _(0-1) — (P3) _(0-2) —P2-NH—CH (CH ₂ C (═O) —NH ₂ ) —C (═O) - or ^{_{R 21 - (C = O)}} (0-1) - (P3) (0-2) -P2-NH-CH (CH 2 COOH) -C (= O) - group or a cathepsin cleavable group R _{^{21 - (C = O) (}} 0-1) - (P3) (1-2) -P2- represent,
R ²¹ represents —H, C _1-10 -alkyl-, C _{5-10 -aryl-} or C _6-10 -aralkyl-, C _5-10 -heteroalkyl-, C _1-10 -alkyl-O—C _{6. -10} - aryl _{-, C 5-10} - heterocyclic alkyl -, heteroaryl -, heteroaryl - alkyl _{-, C 1-10} - alkoxy _{-, C 6-10} - aryloxy - or _{C 6-10} - aralkoxy - , _{C 5-10} - heteroalkoxy _{-, C 1-10} - alkyl _{-O-C 6-10} - aryloxy _{-, C 5-10} - a heterocyclic alkoxy group, it is _-NH 2, -NH- alkyl, -N _{(alkyl) 2, -NH-C (=} O) - alkyl, N (alkyl) -C (= O) - _{alkyl, -SO 3 H, -S (=} O) 2 NH 2, -S (= _O) 2 -N ( _{Alkyl) 2, -COOH, -C (=} O) -NH 2, -C (= O) -N ( _alkyl) may be substituted one or more times with ₂ or -OH, or -H or a group - ( _O) x _- (represents ^{_{CH 2 CH 2 O) y -R}} 22,
x is 0 or 1,
v is a number from 1 to 20,
R ²² is -H, - alkyl (preferably _{C 1-12} - _{alkyl), - CH 2 -COOH, -CH} 2 -CH 2 -COOH, or _{-CH 2 -CH} 2 -NH 2) represents;
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids; when there are a plurality of amino acids P3, P3 can have the same or different amino acids as defined above;
Or R ² and ^{R 4} together (pyrrolidine ring _formation), - CH 2 -CHR ¹⁰ - or ^-CHR 10 -CH ₂ - represents,
R ¹⁰ represents —H, halogen (preferably —F), —NH ₂ , —SO ₃ H, —COOH, —SH or —OH, and the hydrogen atom of the secondary amino group in the pyrrolidine ring is R ²¹ —C _{(= O) -P3 (0-2)} -P2-NH-CH (CH 2 C (= O) -NH 2) -C (= O) -SIG- may be replaced by;
L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
P2 is selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His An amino acid;
P3 is independently Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or equivalent An N-alkyl amino acid, preferably an amino acid selected from one of N-methyl amino acids;
-SIG is a self-destructing group that gives a free secondary amine upon cleavage of the -C (= O) -SIG bond;
A represents —C (═O) —, —S (═O) —, —S (═O) ₂ —, —S (═O) ₂ —NH— or —C (═N—NH ₂ ) —. ;
R ³ is -L- # 1, -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocyclic alkyl group, preferably -L- # 1 or C _1-10 -alkyl , C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-O—C _6-10 -aryl or C _5-10 -heterocyclic alkyl group 1 to 3 —OH groups, 1 to 3 halogen atoms, 1 to 3 halogenated alkyl groups (each having 1 to 3 halogen atoms), 1 to 3 —O-alkyl Group, 1 to 3 —SH group, 1 to 3 —S-alkyl group, 1 to 3 —O—C (═O) -alkyl group, 1 to 3 —O—C (═ O) -NH-alkyl group, Of three -NH-C (= O) - alkyl groups, one to three -NH-C (= O) -NH- group, one to three -S (= _{O) n} - group 1 to 3 —S (═O) ₂ —NH-alkyl groups, 1 to 3 —NH-alkyl groups, 1 to 3 —N (alkyl) ₂ groups, 1 to 3 —NH _Optionally substituted by ₂ or 1 to 3 — (CH ₂ ) _0-3 Z groups,
-L- # 1 represents a linker, # 1 represents a bond to a binder or derivative thereof,
-MOD is represented as defined above,
n represents 0, 1 or 2;
Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H, — (CH ₂ ) _0-3 —CH (NHC (═O) —CH ₃ ) Z ′, — (CH ₂ ) _0-3 —CH (NH ₂ ) Z ′ or — (CH ₂ ) _0-3 represents Z '
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁵ is _-H, -NH _2, -NO _2, halogen (especially -F, -Cl, -Br), - SH or - _{(CH 2)} represents a _0-3 Z,
Z represents -H, -OY ³ , -SY ³ , halogen, -NHY ³ , -C (O) -NY ¹ Y ² or -C (O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ or — (CH ₂ ) _0-3 Z ′;
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ represents —H, —SO ₃ H, —NH ₂ or —COOH;
R ⁶ and R ⁷ are independently of each other —H, cyano, C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _2-10 -alkenyl, fluoro-C _2-10 -alkenyl, C _{2−. 10} -alkynyl, fluoro- _C2-10 -alkynyl, hydroxy or halogen (especially -F, -Cl, -Br);
R ⁸ represents C _1-10 -alkyl, fluoro-C _1-10 -alkyl, C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl, or optionally substituted oxetane;
R ⁹ represents —H, —F, —CH ₃ , —CF ₃ , —CH ₂ F or —CHF ₂ . ] R ¹ represents -H, -L- # 1 or-(CH ₂ ) _0-3 Z;
-L- # 1 represents a linker and # 1 represents a bond to a binder or a derivative thereof;
Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -C (= O) -NY ¹ Y ² or -C (= O) -OY ³ ;
Y ¹ and Y ² each independently represent —H, —NH ₂ , — (CH ₂ CH ₂ O) _0-3 — (CH ₂ ) _0-3 Z ′ or —CH (CH ₂ W) Z ′. ,
Y ³ represents —H or — (CH ₂ ) _0-3 Z ′;
Z ′ is —H, —NH ₂ , —COOH, —NH—C (═O) —CH ₂ —CH ₂ —CH (NH ₂ ) COOH or — (C (═O) —NH—CHY ⁴ ) _{1− 3} represents COOH,
W represents —H or —OH;
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NH—C (═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ Representation;
R ² and R ⁴ independently of each other represent —L— # 1, —H or —C (═O) —CHY ⁴ —NHY ⁵ ;
-L- # 1 represents a linker, # 1 represents a bond to a binder or a derivative thereof,
Y ⁴ represents linear or branched C _1-6 -alkyl which may be substituted by —NH— (C═O) —NH ₂ , or aryl or benzyl which may be substituted by —NH ₂ Represent,
Y ⁵ is -H or -C (= O) represents the -CHY ⁶ -NH _2,
Y ⁶ represents linear or branched C _1-6 -alkyl;
Or R ² and R ⁴ together (form a pyrrolidine ring) represent -CH ₂ -CHR ^10-
R ¹⁰ is _{-L- # 1, -H, -NH 2} , -SO 3 H, -COOH,
Represents -SH or -OH;
L- # 1 represents a linker, # 1 represents a bond to a binder or a derivative thereof,
A represents -C (= O);
R ³ represents — (CH ₂ ) OH or —L- # 1;
-L- # 1 represents a linker, # 1 represents a bond to a binder or a derivative thereof,
R ⁵ represents -L- # 1 or -H;
-L- # 1 represents a linker, # 1 represents a bond to a binder or a derivative thereof,
One of the substituents ^{R 1, R 2, R 3} , R 4 and ^{R 5} represents a-L-# 1, the composite body according to claim 6, as well as their salts, solvates, solvates Salt and epimer. The complex according to claim 6, wherein R ⁶ and R ⁷ independently of one another represent H, C _1-3 -alkyl or halogen. R ⁸ is _{C 1-4} - alkyl (preferably tert- butyl) represents the complex of claim 6. The complex according to claim 6, wherein R ⁹ represents H. The complex according to claim 6, wherein R ⁶ and R ⁷ represent F.   The complex according to one or more of the preceding claims, wherein the binder or derivative thereof is a binder peptide or protein or a derivative of a binder peptide or protein.   The complex according to one or more of the preceding claims, wherein the complex has two binding sites per binder.   The complex according to one or more of the preceding claims, wherein the complex has four binding sites per binder.   15. A complex according to one or more of claims 12 to 14, wherein the binder peptide or protein represents an antibody or a derivative of the binder peptide or protein comprising an acceptor glutamine side chain that can be recognized by transglutaminase.   16. A complex according to one or more of claims 12 to 15, produced by transglutaminase mediated binding.   17. A complex according to one or more of claims 12 to 16, produced from Streptomyces Mobaraensis using transglutaminase.   The complex according to one or more of the preceding claims, wherein the binder binds to a cancer target molecule.   19. The complex of claim 18, wherein the binder binds to an extracellular target molecule.   20. A complex according to claim 19, wherein the binder is internalized after binding to an extracellular target molecule and is treated intracellularly (preferably with lysosomes) by cells expressing the target molecule.   21. A complex according to one or more of claims 12 to 20, wherein the binder peptide or protein is a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof.   The complex according to one or more of the preceding claims, wherein the binder peptide or protein is an antibody having an acceptor glutamine residue in its heavy chain, suitably in the CH2 domain.   23. The complex of claim 22, wherein the binder peptide or protein is an antibody having an acceptor glutamine residue in its heavy chain at position 295 (Kabat numbering system).   23. The antibody according to claim 22, wherein the binder peptide or protein is an antibody comprising a N297X substitution (where X is an amino acid other than asparagine); even more preferably N297D, N297Q, N297S or N297A, very preferably N297A and N297Q. Complex.   23. The complex according to claim 22, wherein the binder peptide or protein is an antibody comprising N297Q substitution and Q295X substitution (where X is an amino acid other than asparagine), preferably Q295N.   The conjugate of one or more of the preceding claims, wherein the binder peptide or protein is an antibody comprising asparagine at residue 297 which has substantially no N-linked glycosylation.   27. The complex of claim 26, wherein the binder peptide or protein is an antibody produced in a host cell that produces an antibody with amino acid residue N297 and no N-linked glycosylation.   The complex according to one or more of the preceding claims, wherein the binder peptide or protein is an anti-HER2 antibody, an anti-EGFR antibody, an anti-TWEAKR antibody or an antigen-binding fragment thereof.   29. The complex of claim 28, wherein said anti-TWEAKR antibody specifically binds to amino acid D at position 47 (D47) of TWEAKR (SEQ ID NO: 169), preferably anti-TWEAKR antibody TPP2090 and its deglycosylated variants. .   The anti-TWEAKR antibody specifically binds to TWEAKR (SEQ ID NO: 169), preferably amino acid D at position 47 (D47) of the anti-TWEAKR antibody TPP-2090-HC-N297A or TPP-2090-HC-N297Q. 30. The complex according to 28 and 29. The linker L is bound to the glutamine side chain of the binder, 1 to 5 kinesin spindle protein inhibitors are bound to the linker L, and the linker L has the following basic structure (i): iv):
(I)-(C = O) _m -SG1-L1-L2-
(Ii)-(C = O) _m -L1-SG-L1-L2-
(Iii)-(C = O) _m -L1-L2-
(Iv)-(C = O) _m -L1-SG-L2
(Wherein, m is 0 or 1, SG and SG1 are in vivo cleavable groups, L1 represents an organic group that is not cleaved in vivo, and L2 is a coupling group to a binder. The complex according to one or more of the preceding claims having one of the following:   The linker L is bound to the glutamine side chain of the binder, 1 to 5 kinesin spindle protein inhibitors are bound to the linker L, and the in vivo cleavable group SG is 2 to 8 oligos 32. Complex according to claim 31, wherein said complex is a peptide group, preferably a dipeptide group or a disulfide, hydrazone, acetal or aminal, and SG1 is a 2 to 8 oligopeptide group, preferably a dipeptide group. 32. The complex of claim 31, wherein the linker is attached to a glutamine side chain and has the formula:

[Where:
m is 0 or 1;
§ represents binding to the active compound molecule,
§§ represents binding to the binder peptide or protein,
L1 is ^{_{- (NR 10) n - (}} G1) represents o -G3-,
R ¹⁰ represents —H, —NH ₂ or C ₁ -C ₃ -alkyl;
G1 represents —NH—C (═O) — or

Represents;
n is 0 or 1;
o is 0 or 1;
G3 represents an optionally substituted straight or branched hydrocarbon chain having 1 to 100 carbon atoms from a bond or arylene group and / or straight chain and / or branched and / or cyclic alkylene group, Groups -O-, -S-, -S (= O)-, -S (= O) _2- , -NH-, -C (= O)-, -NMe-, -NHNH-, -S (= O) ₂ —NHNH—, —NH—C (═O) —, —C (═O) —NH—, —C (═O) —NHNH— and —N—, —O— and —S—, — S (= O)-or -S (= O) _2- (preferably

) May be interrupted one or more times by one or more of 5 to 10 membered aromatic or non-aromatic heterocycles having 4 or less heteroatoms selected from the group consisting of: when present, it _is -NH-C (= O) -NH 2, -COOH, -OH, -NH 2, -NH-CN-NH 2, sulfonamide, sulfone, optionally substituted by a sulfoxide or sulfonic acid well,
Or the following group:

Represents one of the
Rx is _-H, C 1 -C ₃ - alkyl or phenyl,
L2 represents # ^1- (NH) _p- (C = O) _q- G4-NH- # ² or # ^1- (NH) _p- (C = O) _q- G4-O-NH- # ² ,
p is 0 or 1;
q is 0 or 1;
G4 represents an optionally substituted alkyl or heteroalkyl chain, where appropriate, any carbon of the chain is alkoxy, hydroxyl, alkylcarbonyloxy, -S-alkyl, -thiol, -C (= O)- S- alkyl, -C (= O) -NH- alkyl, -NH-C (= O) - alkyl, amine is substituted with -C (= O) -NH _2,
# ¹ represents the point of attachment to the group ^{L 1,}
# ² represents the binding site to the glutamine residue of the binder. ] 34. The complex of claim 33, wherein the hydrocarbon chain is interrupted by one of the following groups.

[ _Wherein X represents H or a C _1-10 -alkyl group, which is —NH—C (═O) —NH ₂ , —COOH, —OH, —NH ₂ , —NH—CN—NH ₂ , sulfone; , May be substituted by sulfoxide or sulfonic acid. ] 35. The complex according to one or more of claims 31 to 34, wherein L2 is one of the following groups.

[Where:
R ^y is —H, —C (═O) —NH-alkyl, —NH—C (═O) -alkyl, —C (═O) —NH ₂ , —NH ₂ ;
# ¹ represents the point of attachment to the group ^{L 1,}
# ² represents the binding site to the glutamine residue of the binder. ] R ^y is -H or -NH-C (= O) complex according to claim 35 is -CH _3. Composite according to claim 35 or 36 R ¹ or ^{R 4} represents a-L-# 1.   The complex according to one or more of the preceding claims, wherein the anti-TWEAKR antibody is an agonist antibody. The binder peptide or protein is
a. CDR1 of the heavy chain encoded by the amino acid sequence comprising the formula PYPMX (SEQ ID NO: 171), where X is I or M;
b. CDR2 of the heavy chain encoded by the amino acid sequence comprising the formula YISPSGGXTHYADSVKG (SEQ ID NO: 172), where X is S or K; and c. CDR3 of the heavy chain encoded by the amino acid sequence comprising the formula GGDTYFDYFDY (SEQ ID NO: 173);
A variable heavy chain comprising: d. CDR1 of the light chain encoded by an amino acid sequence comprising the formula RASQSISXYLN (SEQ ID NO: 174), where X is G or S;
e. A light chain CDR2 encoded by an amino acid sequence comprising the formula XASSLQS (SEQ ID NO: 175), wherein X is Q, A or N; and f. Encoded by an amino acid sequence comprising the formula QQSYXXPXIT (SEQ ID NO: 176) (X at position 5 is T or S, X at position 6 is T or S, and X at position 8 is G or F) CDR3 of light chain
A complex according to one or more of the preceding claims, which is an antibody comprising a variable light chain comprising The binder peptide or protein is
a. A variable sequence of the heavy chain set forth in SEQ ID NO: 10 and further a variable sequence of the light chain set forth in SEQ ID NO: 9, or b. A heavy chain variable sequence set forth in SEQ ID NO: 20 and a light chain variable sequence set forth in SEQ ID NO: 19, or c. A heavy chain variable sequence set forth in SEQ ID NO: 30 and a light chain variable sequence set forth in SEQ ID NO: 29, or d. A heavy chain variable sequence set forth in SEQ ID NO: 40 and a light chain variable sequence set forth in SEQ ID NO: 39, or e. A heavy chain variable sequence set forth in SEQ ID NO: 50, and a light chain variable sequence set forth in SEQ ID NO: 49, or f. A heavy chain variable sequence set forth in SEQ ID NO: 60, and a light chain variable sequence set forth in SEQ ID NO: 59, or g. The variable sequence of the heavy chain set forth in SEQ ID NO: 70, and also the variable sequence of the light chain set forth in SEQ ID NO: 69, or h. The heavy chain variable sequence set forth in SEQ ID NO: 80, and also the light chain variable sequence set forth in SEQ ID NO: 79, or i. A heavy chain variable sequence set forth in SEQ ID NO: 90 and a light chain variable sequence set forth in SEQ ID NO: 89, or j. A heavy chain variable sequence set forth in SEQ ID NO: 100, and a light chain variable sequence set forth in SEQ ID NO: 99, or k. The heavy chain variable sequence set forth in SEQ ID NO: 110 and the light chain variable sequence set forth in SEQ ID NO: 109, or l. 120. A complex according to one or more of the preceding claims, which is an antibody comprising the heavy chain variable sequence set forth in SEQ ID NO: 120 and the light chain variable sequence set forth in SEQ ID NO: 119.   The complex according to one or more of the preceding claims, wherein the antibody is an IgG antibody. The preferably one compound of the following formula in the form of trifluoroacetate salt, is bound to a residue of the binder peptides or proteins using transglutaminase, 2 to 100 times with respect to the compound preferably binder peptides or proteins The method for producing a complex according to one or more of the preceding claims, used in molar excess.

[Wherein X ₁ , X ₂ , X ₃ , SG, L 1, L 3, R 1, R 4, R 10 and R ^y have the same meaning as in claims 1 to 41. ] One compound of the formula

[Wherein X ₁ , X ₂ , X ₃ , SG, L 1, L 3, R 1, R 4, R 10 and R ^y have the same meaning as in claims 1 to 41. ] Complexes according to one of the following formulas: wherein Ak3a, Ak3b, AD3d, Ak3e represent a binder, preferably an antibody, n is 2 to 10, preferably 2 to 4, and more preferably 2 or 4 Represents. ]

45. A complex according to one or more of claims 1 to 44, wherein the binder peptide or protein represents an antibody or a derivative of the binder peptide or protein according to the formula:

  45. A pharmaceutical composition comprising a complex according to claims 1-43 or a compound according to claim 44 or 45 in combination with an adjuvant suitable as an inert and non-toxic medicament.   45. A complex according to one or more of claims 1 to 43 or a compound according to claim 44 or 45 for use in a method for the treatment and / or prevention of diseases.   45. A complex according to one or more of claims 1 to 43 or a compound according to claim 44 or 45 for use in a method for the treatment of hyperproliferative disorders and / or angiogenic disorders.   45. Use of an effective amount of at least one complex according to one or more of claims 1 to 43 or a compound according to claim 44 or 45 for hyperproliferative and / or angiogenic disorders in humans and animals. Treatment and / or prevention methods.

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